Sigma binding region of RNA polymerase and uses thereof

ABSTRACT

The invention provides a method to identify inhibitors of the formation of holoenzyme from core RNA polymerase and sigma.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S.application Ser. No. 60/193,116 filed Mar. 30, 2000, under 35 U.S.C. §119(e).

STATEMENT OF GOVERNMENT RIGHTS

[0002] This invention was made, at least in part, with a grant from theGovernment of the United States of America (grant GM 28575 from theNational Institutes of Health). The Government may have certain rightsto the invention.

BACKGROUND OF THE INVENTION

[0003] The RNA polymerase of Escherichia coli is a large, multisubunitenzyme existing in two forms. The core enzyme, consisting of subunits βand β′ and an a subunit dimer, carries out processive transcriptionelongation followed by termination (Helmann et al., 1988). When one of avariety of sigma (σ) factors is added to core, the holoenzyme is formed(Burgess et al., 1969). The σ subunit confers promoter-specific DNAbinding and transcription initiation capabilities to the enzyme (Helmannet al., 1988; Burgess et al., 1969; Gross et al., 1996; Gross et al.,1992). σ⁷⁰ of E. coli was the first a factor to be described andcharacterized (Burgess et al., 1969). Since then, numerous a factorshave been discovered throughout the Eubacterial kingdom, including sixalternative a factors in E. coli. Each σ subunit directs its cognateholoenzyme to start transcription from only those promoters containingDNA sequences specifically recognized by the σ factor. Thus, generally,each a directs transcription initiation from a specific set of promotersto transcribe genes with related functions. This control oftranscription is mediated partially through the competition of theindividual σ factors for the core enzyme and is a major part of globalgene regulation in bacteria (Zhou et al., 1992).

[0004] As the number of identified a factors increased, it becameapparent that they shared several regions of amino acid sequencesimilarity (Helmann et al., 1988; Gribskov et al., 1986; Lonetto et al.,1992), and the function of the conserved regions is of continuinginterest (Waldburger et al., 1994; Dombrowski et al., 1993; Siegele etal., 1989; Gardella et al., 1989; Lesley et al., 1989). Deletionanalysis of σ⁷⁰ identified a segment of the protein that overlapsconserved region 2.1 (residues 361-390) as being necessary andsufficient for core binding (Lesley et al., 1989). A mutation in ahomologous region of Bacillus subtilis σ ^(E) has also been shown toaffect core binding (Shuler et al., 1995). However, recent findings ofcore binding mutations in other conserved and nonconserved regions ofσ³² have led to the idea of multiple binding sites for the σ subunit onthe core enzyme (Joo et al., 1997; Zhou et al., 1992; Joo et al., 1998;Sharp et al., 1999).

[0005] The β and β′ subunits each contain regions that have highsequence homology with the two largest subunits of eukaryal polymerases(Allison et al., 1985; Sweetser et al., 1987; Jokerst et al., 1989).Some of these conserved regions may act as interaction domains. Aninteraction domain is the minimal region of a protein that canindependently fold to form the secondary and tertiary structure requiredto interact with another protein, DNA, RNA, or ligand. Interactiondomains are larger than the actual binding site which is formed by theamino acids in direct contact with the binding partner. Severinov et al.(Severinov et al., 1992, 1995 and 1996) demonstrated the domain-likeproperties of β and β′ by reconstitution of functional RNA polymerasefrom fragmented β and β′ subunits. Thus, the properties of thepolymerase do not require the entire intact length of the subunit butrather can be generated with smaller domain modules.

[0006] There have been two observations that have identified deletionsin the β or β′ subunits that produce subunits still capable of formingcore enzyme structures but not the holoenzyme. First, a β subunittruncation, missing approximately 200 amino acids of the C terminus, wasshown by glycerol gradient centrifugation to migrate with the other coresubunits but was never seen in the σ-containing fractions (Glass et al.,1986). Second, when immunoprecipitation assays were performed usingreconstituted RNA polymerase containing β′ deletion mutants missingamino acids 201-477, the core subunits were recovered in the samefraction but lacked σ (Luo et al., 1996). However, it was unclearwhether the β′ deletion was non-specific, e.g., prevented correctformation of the interaction domain.

[0007] The idea that a binding is affected by perturbations of the Cterminus of β and the N terminus of β′ is consistent with experimentsshowing that these two subunit termini are physically close together andcan be fused through a flexible linker and still form a functionalenzyme (Severinov et al., 1997). Recent protein-protein footprintingdata have identified a similar region on β′ and two new sites on β forpossible interactions with the σ⁷⁰ subunit (Owens et al., 1998). WhileOwens et al. showed that residues 228-461 of β′ are physically close toσ, the authors did not conclude that there is a direct interactionbetween β′ and σ.

[0008] Burgess et al. (1998) report that residues 260 to 309 of β′ bindto a based on the use of in vitro far-Western and co-immobilizationassays. However, in vitro cell-free binding results do not evidence thatthe region involved in binding in vitro is involved in binding in vivo.For example, it is possible that this region of β′ is buried in thenative structure, e.g., a hydrophobic region, and so would not play αrole in vivo binding. Structural analysis programs indicate thatβ′₂₆₀₋₃₀₉ has two a helices joined by a random coil, and that these twohelices are amphipathic and have the potential for coiled coilformation, based on a heptad repeat motif (Chao et al., 1998; Cohen etal., 1986; Lupas et al., 1991). In particular certain positions known asa and d in the coiled coil motif are hydrophobic and so may be buried innative β′

[0009] Thus, what is needed is the identification of a region in thesubunits of core RNA polymerase that interacts with a in vivo. What isalso needed is a method to identify specific inhibitors of the bindingof a to core RNA polymerase.

SUMMARY OF THE INVENTION

[0010] The invention provides an isolated and purified β′ subunit of RNApolymerase or a portion (i.e., fragment) thereof which specificallybinds to σ in vivo. Preferably, the portion comprises at least 39, morepreferably at least 44, and even more preferably at least 49, residuesof the β′ subunit, although smaller fragments which specifically bind toa in vivo are also envisioned. Also preferably, the isolated andpurified portion of the β′ subunit comprises residues 270 to 309, andeven more preferably residues 260 to 309. As described hereinbelow, aregion on the β′ subunit of RNA polymerase was identified that interactsdirectly with σ (the interaction domain). The in vitro interactiondomain of the β′ subunit with σ was identified by far-Western blotanalysis, which is a general method for mapping a domain on one proteinthat is necessary for binding another protein, and a co-immobilizationassay. As used herein, an “interaction domain” refers to the minimalregion of a protein that can independently fold to form the secondaryand tertiary structures required to interact with another protein, DNA,RNA or ligand. The a binding region of β′ was found to interact withvarious a factors, including σ⁷⁰ and several other E. coli σ's, T4 phageσ gp 55, and σ^(A) from Bacillus subtilis.

[0011] As also described hereinbelow, proteins were prepared which hadsingle point mutations in the predicted coiled coils located withinresidues 260-309 of β′. Several of the mutants were defective forbinding σ⁷⁰ in vitro. Of these mutants, three (R275Q, E295K, and A302Dwhich are change-of-charge mutants at the e and g residues of theβ′₂₆₀₋₃₀₉ predicted coiled coil) were completely defective for growth inan in vivo assay where the mutant β′ is the sole source of β′ subunit.All of the mutants were able to assemble into the core enzyme, however,R275Q, E295K, and A302D were defective for Eσ⁷⁰-holoenzyme formation.Several of the mutants were also defective for holoenzyme assembly withvarious minor a factors. Some mutations were nonfunctional in some ofthe assays but functional in others, indicating that binding of othersites may compensate for loss of binding at the β′₂₆₀₋₃₀₉ site. Thus,these results showed that residues 260 to 309 of the β′ subunitspecifically bind to a in vivo, and that mutations in this region cangreatly diminish core binding of σ⁷⁰ and other minor σ's. In therecently published crystal structure of Thennus aquaticus core RNApolymerase (Zhang et al., 1999), the region homologous to β′₂₆₀₋₃₀₉ ofE. coli forms a coiled coil. Modeling of the β′ mutations describedherein onto that coiled coil places the most defective mutations on oneface of the helix, which may indicate where most of the contact surfacewith σ⁷⁰ occurs. As RNA polymerase is a large multi-subunit complex(having about 3300 amino acids), and the β′ subunit of RNA polymerase isa large protein, e.g., the β′ subunit of E. coli is about 155,000daltons, the identification of the region of the core RNA polymerasewhich specifically interacts with a in vivo represents a significantfinding as it provides a specific target for drug discovery, e.g., drugswhich specifically interfere with the core-σ interaction.

[0012] Thus, the invention provides a method to identify an agent whichinhibits or prevents the binding of σ to core RNA polymerase, a subunitthereof or a portion of the subunit. The method comprises contacting theagent with core RNA polymerase, e.g., isolated core RNA polymerase, oran isolated subunit of RNA polymerase or a portion thereof so as to forma complex. As used herein, “isolated and/or purified” refers to in vitropreparation, isolation and/or purification of a protein or a complex ofbiomolecules, e.g., core RNA polymerase, so that it is not associatedwith in vivo substances or is substantially purified from in vitrosubstances. Preferably, the portion of the subunit comprises at least 39amino acids, more preferably at least 44 amino acids, even morepreferably at least 49 amino acids, of the β′ subunit. The complex isthen contacted with a or a portion thereof and it is determined whetherthe agent inhibits or prevents the binding of a to core RNA polymerase,the isolated subunit of RNA polymerase or portion thereof. A portion ofa comprises at least 30, preferably at least 55, more preferably atleast 100, and even more preferably at least 140 residues of σ, althoughsmaller fragments which specifically bind to β′ in vivo are alsoenvisioned. Alternatively, the agent is contacted with the core RNApolymerase, a subunit and or portion thereof and σ or a portion thereof,i.e., simultaneously. The a may be a homologous a, for example, if thecore RNA polymerase or the isolated subunit of RNA polymerase is that ofE. coli, the σ is a σ which is encoded by the genome of E. coli.Alternatively, the a may be a heterologous a, e.g., a phage-encoded σ.

[0013] Further provided is a method which comprises contacting the agentwith isolated a or a portion thereof so as to form a complex. Thecomplex is then contacted with isolated core RNA polymerase, or anisolated subunit of RNA polymerase or a portion thereof and it isdetermined whether the agent inhibits or prevents the binding of σ tocore RNA polymerase, the isolated subunit of RNA polymerase or portionthereof.

[0014] To find new inhibitors of bacterial transcription, a homogenousluminescence resonance energy transfer (LRET) based assay was developedon the basis of the fluorescent-labeled proteins σ⁷⁰ and β′-fragment(residues 100-309). For the assay, was labeled with a europium chelate(Eu(IJI)-DTPA-AMCA-maleimide) as the LRET donor and β′ was labeled withIC5-maleimide as the acceptor. Measuring time-resolved fluorescence withthe labeled proteins permitted the monitoring of binding of σ⁷⁰ to β′ byobserving the emission of the LRET acceptor (IC5-labeled β′-fragment).The emission of the acceptor is sensitized by an energy transfer fromthe LRET donor (DTPA-AMCA-Eu-complex-labeled σ⁷⁰) that occurs when thedyes come into close proximity to each other (<75 Å). Due to itsnaturally short lifetime of several nanoseconds, the residualICS-fluorescence acquired after 50 microseconds is due solely to LRET,reducing the background signal to a minimum and hence providing a goodsignal-to-noise ratio. The assay was used to measure the effect of theenvironment (solvents, denaturants, and salt) and can be used to measurethe effect of potential inhibitors on the binding of σ⁷⁰ to theβ′-fragment. Such an assay is particularly well suited forhigh-throughput screening.

[0015] Also provided is a method to identify a region on a subunit ofcore RNA polymerase which specifically binds a. The method comprisescontacting core RNA polymerase, e.g., isolated core RNA polymerase, anisolated subunit thereof or a portion thereof with ay or a portionthereof so as to form a complex. The core RNA polymerase, isolatedsubunit or portion thereof comprises at least one amino acidsubstitution. Then complex formation is detected or determined and, forexample, compared to complex formation between core RNA polymerase, anisolated subunit or portion thereof, which does not comprise an aminoacid substitution, and a or a portion thereof.

[0016] The invention further provides a method to identify an agentwhich inhibits or prevents the binding of a to the β′ subunit of coreRNA polymerase. The method comprises contacting a prokaryotic cell withthe agent and detecting or determining whether the agent inhibits orprevents the binding of a to the β′ subunit of RNA polymerase in thecell. The cell may be a recombinant cell, i.e., a cell which isaugmented by exogenously introduced nucleic acid, e.g., bytransformation or transduction. Thus, the invention also provides a hostcell comprising a recombinant DNA encoding a β′ subunit of RNApolymerase.

[0017] The invention further provides agents identified by the methodsof the invention and, in particular, agents which inhibit the growth ofprokaryotic cells which are associated with disease, see e.g., ZinsserMicrobiology (17th ed., Appleton-Century-Crofts, NY (1980).

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1. A schematic of the ordered fragment ladder far-Westernmethod. His₆-tagged target protein is cleaved, and the fragments arepurified on a Ni²⁺-NTA column, fractionated on SDS-PAGE, andelectroblotted onto nitrocellulose. The denaturant is washed away fromthe blotted protein fragments, and the interaction domains on fragmentsare allowed to refold. The interaction domains can be identified byprobing with a radioactively-labeled protein. The interaction domain ismapped by identifying fragments that have part of their interactiondomain missing and can no longer bind the probe.

[0019]FIG. 2. Example of an ordered fragment ladder far-Westernanalysis. A) A schematic indicating the position of the chemicalcleavage sites on the E. coli RNA polymerase β′ subunit for chemicalcleavage agents hydroxylamine (NH₂OH, “Hyd”) and2-nitro-5-thiocyanobenzoic acid (NTCB). These cleavage sites werepredicted from the amino acid sequence using the MacVector program(Oxford Molecular Group). The numbers refer to the amino acid positionsfrom the N-terminus at the left. A “1” indicates the position of acleavage site, while a “2” indicates two sites very close together. B)The ordered fragment ladder of - and C-terminal His₆-tagged β′ subunitcleaved with NH₂OH or NTCB. On the left is shown a schematic of theexpected bands on a SDS gel. In the middle is the actualcoomassie-stained gel, and on the right is an identical gel blotted ontonitrocellulose and probed with ³²P-labeled σ⁷⁰. It can be seen from theNH₂OH cleavage fragment results that most of the N-terminal His₆-taggedfragments bind the probe, while only the full-length C-terminalHis₆-tagged protein binds the probe. Thus, the interaction domain iswithin the region from amino acid 1 to 309 of β′.

[0020]FIG. 3. The β′₂₆₀₋₃₀₉ region. (A) Schematic diagram of β′₂₆₀₋₃₀₉interaction domain. The lettered boxes represent the conserved regionsof eukaryal and prokaryal RNA polymerase largest subunits (Jokerst etal., 1989). β′₂₆₀₋₃₀₉ interaction domain overlaps part of the β′ subunitconserved region B. Below the interaction domain are diagrams of thepredicted a helices and coiled coils. (B) Helical wheel drawing ofpredicted β′₂₆₀₋₃₀₉ coiled coil. The two predicted helices are shown asinteracting with one another to form an antiparallel coiled coil.Mutations are shown next to original residues along with the residuenumber. The N-terminus is at amino acid N266 on the right helix. Thishelix is depicted as coming out of the page, while the left helix isgoing into the page and terminates at N309.

[0021]FIG. 4. Western and far-Western blots of cell extracts containingwild type or mutant β′₁₋₃₁₉. Cell extracts were analyzed by 8-16%Tris-glycine SDS-PAGE, blotted to nitrocellulose, and probed with (A)anti-β′ antibody, or (B) ³²P-labeled σ⁷⁰. (C) Relative binding of σ⁷⁰ bywild type and mutant β′ fragments. The values for relative σ⁷⁰ bindingby wild type versus mutant β′₁₋₃₁₉ fragments as determined from farWestern blotting analysis were normalized to the amount of β′₁₋₃₁₉fragment loaded as determined by quantitative Western blot analysis(wild type=1.0). Error bars represent standard deviation. Results arethe average of three different experiments.

[0022]FIG. 5. Growth with plasmid-derived wild type or mutant β′ as thesole source of β′ subunit. Strain RL602 was transformed with a plasmidencoding either wild type or mutant, full length β′. Transformed cells(10 μI) were then spotted onto duplicate plates, incubated at either 30°C. (permissive) or 42° C. (nonpermissive) for 24-48 hours, and thenassessed for growth.

[0023]FIG. 6. Assembly of core and/or holoenzyme. Cells grown with wildtype or mutant β′ expression plasmids were harvested and subjected topurification to isolate the plasmid-derived His₆-tagged β′ and any ofits assembled complexes. Proteins from Ni²⁺-NTA purified samples wereseparated via SDS-PAGE and blotted to nitrocellulose. The blots werethen probed with monoclonal antibodies (MAbs) against the indicatedsubunits. (A) Log phase samples. (B) Stationary phase samples. No His₆:strain expressing plasmid-derived, wild type β′ without a hexahistidinetag. (C) Quantitation of relative C⁷⁰ binding for the mutants versuswild type β′, normalized to the amount of the a subunit retained (wildtype=1.0). Results are the average of three different experiments. Errorbars represent standard deviation. (D and E) Log and stationary samples,respectively, probed for minor σ factors.

[0024]FIG. 7. Summary of data for mutants.

[0025]FIG. 8. Modeling of mutations. (A) Protein sequence alignment ofE. coli β′₂₆₀₋₃₀₉ and the homologous region from T. aquaticus. Shadedletters represent those not identical to E. coli. (B and C) Two views ofmutations modeled onto the crystal structure of T. aquaticus RNApolymerase (Zhang et al., 1999) using Rasmol software program (Sayle etal., 1995). (B) Looking down center of coiled coil toward polymerase.(C) Side view of coiled coil. The mutations that were defective in allassays tested are colored green. Mutations that were defective in someassays, but not all, are colored cyan. Mutations that were alwaysfunctional are colored purple. “Rudder”, colored maroon, is added toorient the structure.

[0026]FIG. 9. The structures of 6⁷⁰ and the β′-fragment used in theprotein binding assay described in Example 4 are shown. Next to thenames are the mutations and in bold letters the derivatization sites,respectively. HMK-His₆-β′-100-309 is a fragment of β′ which containsresidues 100-309 and is a N-terminal fusion to a heart-muscle kinase(HMK) recognition site and a His₆-tag. The coiled coil α-helicalstructure colored magenta in the β′-fragment is likely the main Gbinding element in the RNAP. IC5 is colored in pink. The regions in σ⁷⁰responsible for binding to core RNAP are regions 2.1 (green) and 2.2(yellow). Also indicated by color are regions 2.3 (blue), 2.4 (brown),the non-conserved region (white) and the N-terminus (red) of theσ⁷⁰-structure. The Eu-DTPA-AMCA complex is colored in dark blue.

[0027]FIG. 10. Dyes used to derivatize the proteins and serve as thefluorophores in the LRET assay.

[0028]FIG. 11. A schematic showing how the LRET signal is created uponbinding of the labeled proteins β′-100-309 and σ⁷⁰. The fluorescence ofthe IC5-labeled β′-fragment decays during the delay of the dataacquisition 50 microseconds after excitation at 320 nm. Only theEu-emission of labeled σ⁷⁰ and the sensitized IC5 emission in thecomplex can be observed after the delay due to the long Eu-luminescenceof over 1 milliseconds. This minimizes the background signal and yieldsin a favorable signal-to-noise ratio that is desired in an efficienthigh-throughput screening assay.

[0029]FIG. 12. Map of the plasmid pTA133. The plasmid pTA133 is derivedfrom the expression vector pET28b(+) (Novagen). An N-terminal HMK-siteand the His₆-tag encoding sequence were inserted together with theβ′-region 100-309. The plasmid carries a pBR322 origin of replicationfor cloning in E. coli and a kanamycin resistance gene for selection.The lacl repressor gene is included for tight control of induction viaIPTG.

[0030]FIG. 13. Plasmid map of the expression plasmid pSigma70(442C). Theplasmid is derived from pGEMX- 1 (Promega) and allows expression under aT7 expression system with ampicillin selection.

[0031]FIG. 14. SDS-PAGE gels of inclusion-body purification forβ′-fragment (left) and σ⁷⁰ (right). Gels were stained with GELCODE(Pierce) Coomassie Blue. Both gels were NuPAGE (NOVEX), with 12%polyacrylamide (left gel) and a gradient of 4-12% polyacrylamide (rightgel).

[0032]FIG. 15. SDS-PAGE gel of the β′-purification and derivatizationsteps (Coomassie stain and IC5 scan). The IC5-scan was performed with aMolecular Dynamics Storm system in the red fluorescence mode.

[0033]FIG. 16. The result of two EMS assays and three differentacquisition methods. On the right side, the Coomassie-stained gel showsthat increasing amounts of labeled β′-fragment can shift all the a intothe upper band representing the complex. The picture on the same gelbelow was acquired with a UV-box, which can only visualize theEu-emission due to the excitation wavelength (312 nm) and an orangefilter on the camera. It confirms that both bands contain Eu-labeled σ.At the bottom of each side is another picture of the same gel as above,this time taken with a Storm imager (Molecular Dynamics) that can onlyvisualize the IC5-label. It confirms that only the upper band containslabeled β′-fragment. The free β′-fragment runs as a diffuse band barelymigrating into the gel (data not shown).

[0034]FIG. 17. Competition of labeled do binding to the β′-fragment byincreasing amounts of unlabeled σ⁷⁰. The decreasing signal is due to thefact that labeled β′-fragment is competed away from the labeled σ⁷⁰ thatit cannot be sensitized via LRET anymore.

[0035]FIG. 18. Dependence of NaCl concentration on the LRET assay. Withincreasing amounts of salt, the LRET signal significantly decreaseswhich should be due to the decreased amount of σ⁷⁰/β′-complex formed.

[0036]FIG. 19. Effect of DMSO concentration in the assay. Increasingamounts of DMSO (0-5%) mixed with the assay buffer previous to theaddition of proteins, do not have a significant effect on the signal.

[0037]FIG. 20. A) Schematic of structural and functional regions of E.coli σ ⁷⁰. B) Sequence alignment of σ⁷⁰ region 2.1-2.2 homologs. C)Sequence alignment of E. coli σ's. D) Sequence alignment of β′ 260-309from various organisms.

DETAILED DESCRIPTION OF THE INVENTION

[0038] Bacterial RNA polymerase synthesizes RNA from cellular genes andthus plays a central role in the regulation of gene expression. Core RNApolymerase can synthesize RNA but is unable to specifically bind DNA atpromoter sites. To specifically bind DNA and initiate transcription ofgenes, a binds to core to form the holoenzyme. A drug which prevents obinding to core polymerase would prevent cell growth. In vitro methods(Example 2 and Burgess et al., 1998) identified a 49 amino acid regionof the β′ subunit of core polymerase which is important for a bindingand which is highly conserved in bacterial polymerases. However, toestablish that this region is important for a binding in vivo,mutational studies were necessary (Example 3). Once the region in coreRNA polymerase which binds to a in vivo is identified, theidentification of broad-range antibiotics, e.g., small molecules such aspeptides or other molecules, which specifically interfere with thebinding is greatly facilitated.

[0039] The invention will be further described by the followingnon-limiting examples.

EXAMPLE 1 Far-Western Blot Mapping

[0040] Transferring materials out of SDS gels (blotting) onto anitrocellulose membrane has become a widely used technique. It not onlytakes advantage of the high resolving power of polyacrylamide gelelectrophoresis but also allows ready access to the blotted targetmaterial by a variety of interaction probes. A Western blot is generallyprobed or detected with an antibody, while a south-Western blot isprobed with a labeled DNA. In a far-Western blot, instead of probingwith an antibody, one probes with another protein, taking advantage ofspecific protein-protein interactions. This approach requires that atleast some region (the interaction domain) of a fraction of the blottedtarget protein be able to refold on the membrane and form a3-dimensional structure containing the interaction site. This approachis particularly useful in determining which subunit of a multisubunitcomplex is involved in an interaction with the probe protein.

[0041] The use of an ordered fragment ladder far-Western analysis takesadvantage of the ease of constructing hybrid proteins containing a tag,for example, a hexahistidine tag (His₆-tags), at either terminus.His₆-tagged proteins bind to Ni-chelate columns, even in the presence ofdenaturants. This method is potentially applicable to anyprotein-protein interaction study. A schematic that illustrates theprinciple of this method using a His₆-tag is shown in FIG. 1.

[0042] Ordered fragment ladder far-Western analysis includes:

[0043] A. cloning and purification of a protein of interest with aHis₆-tag fused to either the N-terminus or C-terminus of that protein;

[0044] B. chemical or enzymatic partial cleavage of the His6-taggedprotein to create a series of fragments;

[0045] C. purification of His₆-tagged fragments on a Ni-chelate affinitycolumn under denaturing conditions to obtain a set of fragments allcontaining the His₆-tagged end;

[0046] D. fractionating these fragments on the basis of size bySDS-polyacrylamide gel electrophoresis to form an ordered fragmentladder;

[0047] E. transferring the protein fragments out of the gel and onto anitrocellulose membrane and allowing these protein fragments to refoldon the membrane;

[0048] F. preparing a ³²P-labeled protein probe, e.g., by labeling aheart muscle protein kinase (HMK) recognition site-tagged protein withy³²P-ATP and heart muscle protein kinase;

[0049] G. probing the membrane with the labeled probe, washing anddetection; and

[0050] H. validating and characterizing the far-Western complex.

[0051] A. Cloning and Purification of Protein with a His₆-tag

[0052] Standard cloning methods are used to place the gene of interestinto an overproducing vector that results in the addition of a His₆-tagon either the - or C-terminus of the protein. A variety of such vectorsare available, e.g., pET vectors (Studier et al., 1990) are a T7polymerase-based expression system available from Novagen (Madison,Wis.). The appropriate overexpression strain is induced withisopropyl-β-D-thiogalactopyranoside (IPTG), the cells lysed, andinclusion bodies prepared (Arthur and Burgess, 1998). The resuspendedinclusion bodies are aliquoted into 1 mg portions and frozen at −70° C.until use.

[0053] B. Chemical and Enzymatic Cleavage of Proteins

[0054] To predict the chemical and enzymatic cleavage sites of anoverproduced, His₆-tagged target protein, the amino acid sequence of atarget protein can be entered into a computer program that predictsprotein cleavage sites (e.g., there are such programs as part of theMacVector or DNA STAR packages). Based on the predicted pattern ofcleavage, one or more cleavage protocols are selected. An example of thepredicted cleavage sites for Escherichia coli RNA polymerase β′ subunitfor two chemical cleavage agents is shown in FIG. 2A.

[0055] Cleavage Protocols

[0056] The conditions described below are exemplary and can be varied,by varying the time of cleavage or the amount of the cleavage reagent,to accommodate proteins that are particularly easy or difficult tocleave. Preferably, cleavage conditions are employed which result in aseven as possible a distribution of fragments. This often is a reactionthat leaves 10-30% of the polypeptide uncleaved.

[0057] Iodosobenzoic Acid Cleavage (Fontana et al., 1983) (cleaves afterTrp)

[0058] 1) Dissolve 1 mg protein in 200 μl 8 M GuHCl.

[0059] 2) Add 800 lil 100% acetic acid, 3 μl p-cresol, and 2 mgiodosobenzoic acid (IBA; Sigma Cat. # 1=8000).

[0060] 3) Incubate at room temperature for 20 hours.

[0061] 4) Speed Vac to dryness (about 1 hour).

[0062] 5) Resuspend in 1 ml urea buffer (Buffer B+8 M urea; Buffer B is20 mM Tris-HCl, pH 7.9; 500 mM NaCl; 5 mM imidazole (Fisher Catalog #BP305-50); 0.1% (v/v) Tween 20; and 10% (v/v) glycerol).

[0063] 6) Load on Ni-column.

[0064] 2-nitro-5-thiocyanobenzoic acid (NTCB) Cleavage (Jacobson et al.,1973) (cleaves before Cys)

[0065] 1) Dissolve 1 mg protein in 1 ml urea buffer without glycerol.

[0066] 2) Add 5-fold molar excess (to Cys in protein) of dithiothreitol(DTT) made fresh (1 M stock).

[0067] 3) Incubate 15 minutes at 37° C. to reduce disulfides.

[0068] 4) Add 5-molar excess (over total Cys) NTCB (Sigma Cat. # N-7009)and adjust pH to 9.5 with NaOH.

[0069] 5) Incubate at room temperature for 2-6 hours for partialcleavage or 24-30 hours for total cleavage.

[0070] 6) Dilute 1:10 in urea buffer and load on Ni-column.

[0071] Hydroxylamine Cleavage (Bornstein and Bolian 1970) (cleavesbetween Asn-Gly)

[0072] 1) Dissolve 1 mg protein in 1 ml urea buffer.

[0073] 2) Incubate 15 minutes at 37° C.

[0074] 3) To 500 IIl of urea-solubilized protein, add 500 μl ofhydroxylamine buffer (400 mM CHES buffer pH 9.5; 4 Mhydroxylamine-hydrochloride (Aldrich Cat. No. 15,941-7); and adjust pHto 9.5 with 10 M NaOH) and incubate 2 hours at 42° C.

[0075] 4) Add 7 μl 2-mercaptoethanol (to 0.1 M), mix, and incubate for15 minutes at 37° C.

[0076] 5) Dilute 1:10 in urea buffer and load on Ni-column.

[0077] Thermolysin Cleavage (Rao et al., 1996) (cleaves beforehydrophobic amino acids)

[0078] 1) Resuspend 1 mg of inclusion body protein in 100 μl of ureabuffer.

[0079] 2) Incubate for 15 minutes at 37° C.

[0080] 3) Add thermolysin (from Bacillus thennoproteolyticus, BoehringerMannheim) at protein:protease ratios of 4,000:1, 8,000:1 and 16,000:1(w/w).

[0081] 4) Digest for 30 minutes at room temperature.

[0082] 5) Load on Ni-column.

[0083] Trypsin Cleavage (Rao et al., 1996) (cleaves after Arg and Lys)

[0084] 1) Resuspend 1 mg of inclusion body protein in 1 ml of ureabuffer.

[0085] 2) Incubate for 15 minutes at 37° C.

[0086] 3) Dilute to 4 M urea by adding an equal volume of buffer B.

[0087] 4) Add trypsin (TPCK treated, Worthington Biochemicals) atprotein:protease ratios of 4,000:1, 8,000:1 and 16,000:1 (w/w).

[0088] 5) Digest for 30 minutes at room temperature.

[0089] 6) Load on Ni-column.

[0090] Chemical cleavage ladders are very useful in determining theprecise size of fragments since one knows exactly where the cleavageoccurs. This is particularly important since many proteins migrateabnormally upon SDS polyacrylamide gel electrophoresis.

[0091] Since most chemical cleavage reagents only produce a few cuts perpolypeptide chain, the ordered fragment ladder generated by chemicalcleavage has only a few “rungs” on the ladder. Therefore, partialcleavage with one or more proteases can be used to create a ladder withmore rungs that is capable of higher resolution mapping of interactiondomains. Light, moderate, and heavy cleavage reactions can be performedwith a given protease, and the resulting cleavage reactions mixedtogether before or after purification on the Ni-chelate column. Thishelps to produce a ladder containing similar amounts of each fragmentsize.

[0092] Sometimes it is difficult to generate a good ladder by partialcleavage methods, either because of a scarcity or uneven distribution ofchemical cleavage sites or because the protein is relatively resistantto proteolysis. In these cases one can also produce ladders by cloningindividual truncated fragments.

[0093] C. Ni-Chelate Column Purification of His6-tag Fragments Procedure

[0094] 1) Load Ni²⁺-NTA resin (Ni²⁺-NTA agarose, Qiagen) slurry intoBioRad mini column to generate a 300 μcolumn bed.

[0095] 2) Wash with 5 column volumes of MilliQ water. All columnoperations are carried out at room temperature.

[0096] 3) Wash with 5 column volumes of urea buffer.

[0097] 4) Load cleavage reaction (see above) and let drain to top ofresin.

[0098] 5) Wash with 10 column volumes of urea buffer to removenon-His-tagged fragments.

[0099] 6) Wash with 10 column volumes of buffer B to remove urea.

[0100] 7) Elute with 500 μl buffer B with 200 mM imidazole.

[0101] 8) Check extent of cleavage by SDS-PAGE.

[0102] 9) Store fragments as 50 μl aliquots frozen at −20° C.

[0103] The use of Ni-chelate column purification permits His-taggedproteins or fragments to bind to Ni²⁺-NTA column, even in the presenceof 8 M urea or 6 M GuHCl. Washing with a solution containing adenaturant prevents interactions between hydrophobic protein fragmentsand ensures that only His₆-tagged fragments are purified.

[0104] Once a set of ordered fragments are produced, they may be storedat −20° C. or −70° C. for over a year until they are needed to map thebinding of a monoclonal antibody or interacting protein.

[0105] D. Gel Electrophoresis

[0106] Standard SDS polyacrylamide gel electrophoresis procedures areemployed. Colored MW markers (such as the Novex MultiMark Multi-ColorStandards) may aid in determining if the transfer to nitrocellulose isefficient and to aid in cutting the nitrocellulose filter if probingwith several different radioactive probes or antibodies. Pre-poured8-16% gradient polyacrylamide Tris-glycine gels (Novex) are useful tovisualize both large polypeptides like the E. coli RNA polymerase β′subunit and smaller, e.g., partial proteolysis, fragments on the samegel.

[0107] E. Transfer of Protein Fragments from an SDS Gel to aNitrocellulose Membrane

[0108] The proteins or peptides separated by SDS gel electrophoresis areelectrophoretically transferred to nitrocellulose membrane as describedbelow prior to either Western analysis or far-Western analysis.

[0109] Procedure

[0110] 1) Cut 1 piece nitrocellulose and 2 pieces Whatman paper (3 MMchromatography paper, Fisher cat. #05-714-5) slightly larger than gel.

[0111] 2) Pre-wet 1 sponge and 1 piece Whatman paper with Towbin Buffer(TB) (Towbin et al., 1979) (for 2 liters-400 ml methanol (20% final);500 ml 4×Tris-glycine (1×final; for 1 L of 4×Tris-glycine, pH 8.5-57.6 gglycine and 12.0 g. Tris base); 10 ml 10% SDS (0.05% final).

[0112] 3) Place Whatman paper on top of the sponge, then place the gelon top of paper.

[0113] 4) Wet nitrocellulose (Schleicher & Schuell Protran 0.05 μm, cat.# 00870) and place it on the gel (avoid bubbles between gel andnitrocellulose) followed by wet Whatman paper and 2 sponges.

[0114] 5) Place the resulting sandwich in the cage and put it into thetransfer box with the nitrocellulose membrane towards the positiveterminal.

[0115] 6) Fill the transfer box with TB and transfer for 3 hours at aconstant current of 200 mA (about 60 volts).

[0116] 7) Remove the nitrocellulose, place it protein side up in a petridish, add 10-25 ml of Blotto (2% (w/v) Carnation nonfat dry milk inTBST; for 1 liter of TBST-10 ml of 1 M Tris-HCl, pH 7.9 (10 mM final);37.5 ml of 4 M NaCl (150 mM final); 1 ml Tween 20 (0.1% final)) to coverthe blot, and block the membrane for 1-2 hours at room temperature withshaking or overnight at 4° C.

[0117] 8) For Western analysis: Wash 1×with TBST for about 30 seconds atroom temperature; incubate 1 hour at room temperature in 10 ml with1:1000 dilution of primary antibody; wash 3×with 10 ml TBST for 5minutes each; incubate 1 hour at room temperature in 10 ml Blotto with1:1000 dilution of secondary antibody conjugated with horseradishperoxidase (HP) or alkaline phosphatase (AP); wash 3 times with 10 mlTBST for 5 minutes each; and develop with an appropriate calorimetric orchemiluminescent detection reagent.

[0118] 9) For far-Western analysis: proceed as described in section Gbelow.

[0119] F. ³²p Labeling Proteins with Protein Kinase A for Far-WesternProbing

[0120] If the 5-amino acid recognition site for the catalytic subunit ofthe cAMP-dependent protein kinase A from heart muscle (RRASV) isattached to the terminus of a cloned protein, the protein can readily belabeled by reaction with y³²P-ATP and protein kinase A (Li et al., 1989;Blanar et al., 1992; Destka et al., 1999). A cloning vector was preparedthat was based on the pET vector pET-28b+(Novagen), which contains theHMK recognition site and results in an N-terminal addition to a clonedprotein of 25 amino acids (deArruda and Burgess, 1996). The vector isavailable from Novagen as pET-33b(+). Several additional vectors wereprepared for the purpose of producing HMK site-tagged protein probes.These constructs contain either a NdeI or NcoI cloning site that allowsone to fuse the N-terminal Met of the probe protein to a 13-amino acidN-terminal HMK-His₆ tag and provides the choice of either kan^(R) or anamp^(R) antibiotic resistance marker. The relevant information aboutthese vectors is summarized below in Table 1. TABLE 1 Vector DerivedCloning Resistance Name From N-terminal tag Site Marker pAP1 pET-28bMARRASVHHHHHH (SEQ ID NO:2) NdeI kan^(R) pAP2 pET-21a MARRASVHHHHHH (SEQID NO:3) NdeI amp^(R) pAP3 pET-32b MRRASVHHHHHHA (SEQ ID NO:4) NcoIamp^(R)

[0121] Preparation of HMK recognition site-tagged probe protein

[0122] A BL21 (DE3) E. coli strain (Studier et al., 1990), containingthe probe protein cloned into a suitable vector such as one of thosedescribed above, is cultured, induced and the inclusion bodies purifiedas described in Arthur and Burgess (1998). The washed inclusion body maybe solubilized with GuHCl or the detergent sodium-N-lauroyl sarcosine(Sarkosyl) and refolded as described in Burgess (1996) and Marshak etal. (1996). Often the washed inclusion bodies are solubilized with 8 Murea and purified either before or after refolding by affinitychromatography on a Ni-chelate column (Burgess et al., 1998).

[0123] Procedure

[0124] 1) Add 5 μl of 10X 10×protein kinase A (PKA) Buffer (Pkase Kit,cat. # 70510-3 from Novagen) (200 mM Tris-HCl, pH 8.0, 1.5 M NaCl, 200mM MgCl₂, 100 μM ATP) to a 1.5 ml microfuge tube.

[0125] 2) Add 20-40 μg (about 500 pmol) of protein to be labeled (oftenstored in 50% glycerol) and bring total volume to 43 μl with MilliQwater. The final glycerol concentration should be 20-25%.

[0126] 3) Add 5 μl PKA (Novagen; 20 U/μl stock) and 2 μl γ³²P-ATP(NEN/DuPont, 600 Ci/mmol, 5 mCi/33 μl; 300 μμCi=6.6×10⁸ dmp), mix, andincubate for 60 minutes at room temperature.

[0127] 4) Add 50 μl of 1×Labeling Buffer (1×LB) (25% glycerol; 40 mMTris-HCl, pH 7.4; 100 mM NaCl; 12 mM MgCl₂; 0.1 mM DTT (added fresh)) toreaction, add the resulting 100 μl of diluted reaction to a washed spincolumn (BioSpin P6 from Bio Rad), and spin 4 minutes at 1000 g. Justbefore use, vortex the column to resuspend the resin, remove the columnbottom, and allow the column to drain. Add 1 ml 1×LB and allow it toflow through by gravity. Spin in Beckman TJ-6 centrifuge (TH-4 swingingbucket rotor) in 50 ml conical plastic tube at 1000 g for 2 minutes atroom temperature and discard flow-through.

[0128] 5) Collect flow through in a microfuge tube.

[0129] 6) Store labeled probe frozen at −20° C. until use. It can bestored for up to 30 days.

[0130] Approximately 30-50% of the label is incorporated into protein,and after the spin column, about 90% of the label is in protein. Atypical labeling of HMK-σ⁷⁰ at 35 μg in a 50 μi reaction gives about1-4×10⁶ cpm/μg. The above protocol yields about 100 μl of material,suitable for probing 10-20 far-Western blots. One can also label withγ³³P-ATP (deArruda and Burgess, 1996). While this gives a lower specificactivity, and thus a lower detection sensitivity, it does result in alabeled probe that has a longer half-life and which gives sharper bandson imaging.

[0131] G. Probing Far-Western Blots with ³²P-Labeled Protein Procedure

[0132] 1) Transfer proteins or fragments from gel or spot proteins ontonitrocellulose membrane.

[0133] 2) Block the membrane 2 hours in probe buffer (ProB; final 20 mMHepes, pH 7.2; 200 mM KCl; 2 mM MgCl₂-6H₂O; 0.1 mM ZnCl₂; 1 mM DTT; 0.5%Tween 20; 1% Nonfat dried milk; 10% glycerol; in MilliQ water) at roomtemperature with shaking (or overnight at 4° C.).

[0134] 3) Add 5-10 μl of labeled probe (³²P-labeled protein) solution to15 ml of ProB and incubate for 2 hours with the membrane at roomtemperature with shaking.

[0135] 4) Wash the membrane 3 times for 3 minutes each with 10 ml ProB.

[0136] 5) Air dry the membrane (about 15 minutes), wrap in Saran Wrap,and expose it to film or a Phosphorimager screen (Molecular Dynamics).

[0137] This method is more powerful if one can generate ordered fragmentladders of both - and C-terminally His₆-tagged versions of the targetprotein. That way one can map the interaction domain from bothdirections. FIG. 2B shows a predicted fragment pattern and a coomassieblue-stained SDS gel for the ordered fragment ladders that results fromcleaving both N-terminal (N) and C-terminal (C) His₆-tagged E. coli RNApolymerase β′ subunit with hydroxylamine (NH₂OH) or with NTCB. Theright-hand part of FIG. 2B shows the results of a far-Western analysisof an identical gel, probed with ³²P-labeled σ⁷⁰ (See Example 2).

[0138] Ordered fragment ladder far-Western analysis requires that atleast a fraction of the molecules in a blotted fragment band are able torefold at least that part of the polypeptide (the interaction domain)needed to create the 3-dimensional interaction surface or interactionsite. A number of papers have reported increased refolding and thussensitivity in far-Western analysis when the blotted target protein issubjected to denaturation followed by renaturation prior to probing(Lieberman and Berk, 1991; Vinson et al., 1988). Presumably, the SDStransferred with the blotted protein is removed (by becoming bound tothe large excess of casein in the probe buffer), allowing the targetprotein to refold, at least partially, on the membrane.

[0139] Nitrocellulose pore sizes larger than 0.05 μm can be used,however, 0.05 μm has better retention of small protein fragments.

[0140] Several different labeled probes bind non-specifically to coloredMW markers, most likely an interaction between the His₆-tag or theHMK-tag and the dyes that are attached to the markers. This, however,provides a useful set of labeled markers to help one orient theresulting data.

[0141] H. Validating and Characterizing the Far-Western Complex

[0142] If a positive signal is detected in a far-Western analysis,additional evidence may be necessary to show that the binding observedis due to a specific, relevant interaction and not merely a non-specificionic or hydrophobic interaction. These other methods include theco-immobilization assay (Example 2; Arthur and Burgess, 1998; Burgess etal., 1998), and site-directed mutagenesis of the interaction domain(Example 3). The co-immobilization assay involves cloning the putativeinteraction domain into a vector which links the domain to a His₆-tagand passing the resulting protein over a Ni-chelate column. If the probe(lacking a His₆-tag) binds to the immobilized target domain and elutesfrom the column when the target is eluted with imidazole, then one caninfer that the two proteins interact. Smaller target domains can beobserved in the co-immobilization assay, possibly due to the fact thatthe refolded target domain is attached to the Ni-chelate column throughinteraction of its terminal His₆-tag and so can display the smallestfunctional interaction domain. By contrast, the blotted fragment must beattached by at least one or more contacts between the protein and themembrane. It requires that extra amino acids be present to allow bindingwithout interfering with refolding of the minimal interaction domain onthe membrane.

[0143] Significant non-specific interaction can be ruled out bydemonstrating that the labeled probe does not give a signal abovebackground when used to probe a blot of a number of proteins such as BSAor major bacterial proteins in a bacterial extract.

[0144] The nature of the probe-target complex can be partiallycharacterized in an ordered fragment ladder far-Western analysis. Thiscan be accomplished by probing as described in section G and thenwashing the membrane with probe buffer for varying lengths of time andmeasuring the amount of labeled probe remaining bound to the membrane.In this way, one can determine the approximate half-life of the complexon the membrane. For example, the complexes between σ⁷⁰ and fragments ofβ′ dissociate with half-lives of about 2.5 hours. Similarly, one canvary the salt during such a wash and determine the effect of salt on therate of dissociation.

[0145] Conclusions

[0146] A positive result is useful provided you can show it is specific.This is a rapid means of detecting binding and locating the regioncontaining an interaction domain. It can direct one to focus moretedious mapping approaches, such as cloning individual truncatedfragments or making multiple mutations, to a relatively small segment ofthe target polypeptide. The use of the ³²P-labeled protein probe allowsrelatively weak interactions to be detected. The probe can easily belabeled to over 106 cpm/μg and the final wash after incubation of theblot with the probe and before exposure to PhosphorImaging can be aslittle as 5-10 minutes. In contrast, the detection of bound probe byimmunological methods requires incubation with primary and secondaryantibodies that can take several hours or more. The extended incubationand wash time can allow the probe to dissociate from the target.

[0147] This method does not map the interaction site, but rather thewhole region (the interaction domain) needed to form the contact surfaceor interaction site. An important interaction might not be detected ifthe interaction domain is difficult to refold or binds the probe proteintoo weakly. In order to detect binding, one must be within the “windowof the assay”, i.e., the half-life must be longer than the time of thewash. Convincing results are not obtained if the binding is weak, on theorder of the non-specific binding to random proteins or background. Thismethod is ineffective if the interaction domain involves regions of twodifferent-sized polypeptides and might not work if it involves twodistant regions of the same polypeptide. It also would work poorly ifthere were a strong nitrocellulose membrane-binding site in the middleof the interaction domain that prevented refolding on the membrane.

[0148] This method is useful to map epitopes of antibodies,protein-protein interaction domain mapping, DNA or RNA binding sitemapping, and mapping sites of modification, e.g., sites of radioactivemodification, such as phosphorylation or the labeled tag from atag-transfer cleavable cross-linker (Chen et al., 1994) along apolypeptide. One could start with a His₆-tagged protein, allow themodification to occur, chemically or enzymatically cleave the targetprotein, isolate the His₆-tagged fragments on a Ni-chelate column,fractionate by SDS gel electrophoresis, transfer to a membrane, andexpose to film or a Phosphorlmager screen to determine at which point asyou move down the ordered fragment ladder you no longer detect thelabel.

EXAMPLE 2 Far-Western Blot Analysis of β′, β and σ

[0149] Materials and Methods

[0150] Plasmids

[0151] Plasmid characteristics are described in Table 2. TABLE 2 PlasmidSubunit Residues Modifications Ref. pTA499 β′ 1-1407 N-terminal His₆Arthur and Burgess, 1998 pTA500 β′ 1-1407 C-terminal His₆ Arthur andBurgess, 1998 pTA501 β 1-1342 N-terminal His₆ Arthur and Burgess, 1998pTA502 β 1-1342 C-terminal His₆ Arthur and Burgess, 1998 pTA515 β′ 1-260None Arthur and Burgess, 1998 pTA516 β′ 1-280 None Arthur and Burgess,1998 pTA517 β′ 1-300 None Arthur and Burgess, 1998 pTA518 β′ 1-309 NoneArthur and Burgess, 1998 pTA519 β′ 150-309 None Arthur and Burgess, 1998pTA522 β′ 1-260 N-terminal His₆-HMK Arthur and Burgess, 1998 pTA523 β′1-280 N-terminal His₆₋HMK Arthur and Burgess, 1998 pTA524 β′ 1-300N-terminal His₆-HMK Arthur and Burgess, 1998 pTA525 β′ 1-309 N-terminalHis₆₋HMK Arthur and Burgess, 1998 pTA528 β′ 60-309 None Arthur andBurgess, 1998 pTA530 β′ 100-309 None Arthur and Burgess, 1998 pTA531 β′33-309 N-terminal His₆-HMK Arthur and Burgess, 1998 pTA532 β′ 60-309N-terminal His₆-HMK Arthur and Burgess, 1998 pTA533 β′ 100-309N-terminal His₆-HMK Arthur and Burgess, 1998 pTA534 β′ 150-309N-terminal His₆-HMK Arthur and Burgess, 1998 pTA535 β′ 178-309 NoneArthur and Burgess, 1998 pTA536 β′ 200-309 None Arthur and Burgess, 1998pTA537 β′ 178-309 N-terminal His₆-HMK Arthur and Burgess, 1998 pTA538 β′200-309 N-terminal His₆-HMK Arthur and Burgess, 1998 pTA540 β′ 260-1407C-terminal His₆ Arthur and Burgess, 1998 pTA546 β′ 260-309 C-terminalHis₆ Arthur and Burgess, 1998 pTA547 β′ 270-1407 C-terminal His₆ Arthurand Burgess, 1998 pTA548 β′ 280-1407 C-terminal His₆ Arthur and Burgess,1998 pTA549 β′ 290-1407 C-terminal His₆ Arthur and Burgess, 1998 pRL663β′ 1-1407 C-terminal His₆ Wang et al., 1995 pRL706 β 1-1342 C-terminalHis₆ Severinov et al., 1999 pHMK- α⁷⁰ 1-613 N-terminal His₆-HMK Arthurand His₆-α⁷⁰ Burgess, 1998 pLN12 α⁷⁰ 1-613 None Rao et al., 1996

[0152] Construction of Plasmids

[0153] An overexpression vector for C-terminal hexahistidine(His₆)-tagged β′ (pTA500) was constructed by removing the XbaI-HindfIfragment from pRL663 and placing it in pET28b (Novagen) (Studier et al.,1990). N-terminally His₆-tagged A was expressed from pTA499 that wasconstructed using PCR to place the His₆ tag on the N terminus of afragment that overlapped the NruI site of β′. This fragment was placedinto the pET28b vector followed by the insertion of the C-terminalportion of the gene on a NruI-HindlIH fragment from pRL663. TheC-terminal His₆ tag from the pRL663 fragment was removed by replacementof the RsrII-HindIII fragment with a PCR product coding for thewild-type C terminus. pTA501 was constructed by creating an N-terminalHis₆ tag via PCR for a fragment of β, which overlapped the KpnI site ofp. The fragment was placed into the pET28b vector. The C terminus of thegene was added by insertion of a KpnI-Hindfi fragment containing thewild type coding sequence. pTA502, coding for the C-terminal His₆-taggedβ subunit, was derived using PCR to insert a N-terminal NcoI site onto afragment overlapping KpnI. The C-terminal His₆-containing fragment wasinserted on a KpnI-HindIR fragment from pRL706 (Severinov et al., 1997).

[0154] Vectors expressing unmodified fragments of β′ were obtained byPCR cloning of the desired fragment and placement of the fragment intoeither pET21 a (Novagen) for pTA528, pTA530, pTA535, and pTA536 orpET24a (Novagen) for pTA519 using NdeI and XhoI restriction sites.pTA522-525, pTA53 1, and PTA533 were all created by amplifying thespecified β′ region via PCR and inserted into a pET21 a derivative thathad been modified to fuse a N-terminal His₆ and heart muscle kinase(HMK) recognition site to the expressed proteins. pTA532 and pTA534 wereconstructed in the same fashion with the exception that the His₆-HMKvector derivative was constructed from pET28b. pTA547-549 were createdby inserting the fragments, N-terminally truncated via PCR, thatoverlapped the SnaBI site of β′ into pET24a. The C-terminal codingregion of the gene was inserted on a SnaBI-HindlMl fragment from pTA500.pTA546 was created by fusing a C-terminal His₆ tag directly afterresidue 309 via PCR. The fragment was placed into the pET24a vectorusing NdeI and XhoI sites. To use σ⁷⁰ as a radioactive probe, the HMKsite was fused to the N terminus of 070 along with a His₆ purificationtag. pHMK-His₆-σ⁷⁰ was created by placing the σ⁷⁰ gene into a derivativeof pET28b vector that contained the N-terminal His₆ and HMK fusion andadds a total of 13 extra amino acids (MHHHHHHARRASV; SEQ ID NO:5) to theN terminus of σ⁷⁰. All products created by PCR were sequenced to ensurethat no mutations had been introduced.

[0155] Expression and Purification of Proteins

[0156] Plasmids were transformed into BL21(DE3) (Novagen) forexpression. The cells were grown in 1 liter cultures at 37° C. in LBmedium with either 100 μg/ml ampicillin or 50 jig/ml kanamycin. Thecultures were grown to an A₆₀₀ between 0.6 and 0.8 and then induced with1 mM isopropyl-β-D-thiogalactopyranoside. Three hours after induction,the cells were harvested by centrifugation at 8,000×g for 15 minutes andfrozen at −20° C. until use.

[0157] The cells were thawed and resuspended in 10 ml of lysis buffer(40 mM Tris-HCl, pH 7.9, 0.3 M KCl, 10 mM EDTA, and 0.1 mMphenylmethylsulfonyl fluoride), and lysozyme was added to 100 μg/ml. Thecells were incubated on ice for 15 minutes then sonicated three times in60 second bursts. The recombinant protein in the form of inclusionbodies was separated from the soluble lysate by centrifugation at27,000×g for 15 minutes. The inclusion body pellet was resuspended, bysonication, in 10 ml of lysis buffer +2% (w/v) sodium deoxycholate. Themixture was centrifuged at 27,000×g for 15 minutes and the supernatantdiscarded. The deoxycholate-washed inclusion bodies were resuspended in10 ml deionized water and centrifuged at 27,000×g for 15 minutes. Thewater wash was repeated, and the inclusion bodies were aliquoted into 1mg pellets and frozen at −20° C. until use.

[0158] σ⁷⁰ inclusion bodies were solubilized, refolded, and purifiedaccording to a variation of the procedure of Gribskov and Burgess(1983). The inclusion bodies were solubilized by resuspension in 6 Mguanidine HCl (GuHCl). The proteins were allowed to refold by dilutingthe denaturant 64-fold with buffer A (50 mM Tris-HCl, 0.5 mM EDTA, and5% (v/v) glycerol) in 2-fold steps over 2 hours. One gram of DE52 resin(Whatman) was added and mixed with slow stirring for 24 hours at 4° C.The resin was then collected in a 10 ml column, washed, and the proteineluted with a gradient from 0.1 to 1 M NaCl in buffer A. The σ⁷⁰fractions were pooled and dialyzed overnight against 1 liter of storagebuffer (50 mM Tris-HCl, 0.5 mM EDTA, 0.1 M NaCl, 0.1 mM DTT, and 50%(v/v) glycerol) and stored at −20° C.

[0159] Whole cell lysates were prepared as follows. Cells containingtruncated β′ expression plasmids were grown to an A₆00 of 0.6-0.8 andinduced with 1 mM isopropyl-β-D-thiogalactopyranoside. The cells weregrown for an additional 30 minutes. A 200 μl sample was removed andsonicated 3×30 seconds. Twenty μl of glycerol and 20 μl of SDS-samplebuffer were added and heated for 2 minutes at 95° C. then stored at −20°C. until use.

[0160] Protein Cleavage

[0161] β and β′ inclusion bodies were subjected to chemical or enzymaticcleavage (see below) and then purified by nickel affinity chromatographyas follows. The cleavage reaction was loaded onto 300 μl of Ni²⁺-NTAresin (Qiagen) in a Bio-Rad mini-column. The resin had beenpre-equilibrated with buffer B (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 5mM imidazole, 0.1% (v/v) Tween 20, and 10% (v/v) glycerol) +8 M urea.The protein bound resin was washed with 10 column volumes of buffer B+8M urea followed by 10 column volumes of buffer B to allow refolding. Theresin was then washed with 500 ll of buffer B+40 mM imidazole. Theprotein was eluted with 500 μl of buffer B+200 mM imidazole. The elutedfractions were stored at −20° C.

[0162] NTCB Cleavage (Jacobson et al., 1983)

[0163] One mg of inclusion body protein was resuspended in 1 ml ofbuffer B+8 M urea. DTT was added to 5-fold molar excess over the thiolgroups in the protein. The mixture was incubated for 15 minutes at 37°C. to reduce any disulfide bonds. NTCB was added to 5-fold molar excessover total sulfhydryl groups. The pH was adjusted to 9.5 with NaOH. Thereaction mixture was incubated for 2 hours at room temperature. Thecleavage mixture was diluted 1: 10 in buffer B+8 M urea and loaded ontoa Ni²⁺-NTA column as described above.

[0164] Hydroxylamine Cleavage (Bornstein et al. 1970)

[0165] One mg of inclusion body protein was resuspended in 1 ml ofbuffer B plus 8 M urea. Five-hundred microliters of the solubilizedprotein were added to 500 μl of hydroxylamine cleavage solution (0.4 MCHES, pH 9.5, 4 M hydroxylamine HCl) and incubated 2 hours at 42° C.P-Mercaptoethanol was added to 0.1 M and incubated 10 minutes at 37° C.The mixture was diluted 1:10 in buffer B+8 M urea and loaded onto aNi²⁺-NTA column as described above.

[0166] Themolysin Cleavage (Rao et al.. 1996)

[0167] One mg of inclusion body protein was resuspended in 100 μl ofbuffer B+8 M urea and incubated for 15 minutes at 37° C. Thermolysin wasadded at protein:protease ratios of 4,000:1, 8,000: 1, and 16,000:1(w/w). Reactions were carried out for 30 minutes at room temperature.The reactions were loaded onto a Ni²⁺-NTA column as described above.

[0168] Trypsin Cleavage (Rao et al. 1996)

[0169] One mg of inclusion body protein was resuspended in 1 ml ofbuffer B+8 M urea and incubated for 15 minutes at 37° C. The mixture wasdiluted to 4 M urea by adding an equal volume of buffer B. Trypsin wasadded at protein:protease ratios of 4,000:1, 8,000:1, and 16,000:1(w/w). Digestion was performed at room temperature for 30 minutes. Thereactions were loaded onto a Ni²⁺-NTA column as described above.

[0170] Far-Western Blotting

[0171] Dot Blot

[0172] Inclusion body proteins that were resuspended in buffer B+8 Murea were spotted directly onto a nitrocellulose membrane (Schleicher &Schuell) using a Schleicher & Schuell “MINIFOLD” dot blot apparatus. Thewells were washed three times with buffer B. The nitrocellulose wasblocked by incubation in HYB buffer (20 mM Hepes, pH 7.2, 200 mM KCl, 2mM MgCl₂, 0.1 mM ZnCl₂, 1 mM DTT, 0.5% (v/v) Tween 20, 1% (w/v) non-fatdry milk) for 16 hours at 4° C.

[0173] Gel Blot

[0174] Protein cleavage fragments or whole cell lysates were separatedby SDS-polyacrylamide gel electrophoresis (PAGE). The proteins wereelectrophoretically transferred onto 0.05 μm nitrocellulose. Thenitrocellulose was blocked by incubating in HYB buffer for 16 hours at4° C.

[0175] Labeling

[0176] Labeling of σ⁷⁰ was done in a 100-μl reaction volume. Fifty μl of2×kinase buffer (40 mM Tris-HCl, pH 7.4, 200 mM NaCl, 24 mM MgCl₂, 2 mMDTT, and 50% (v/v) glycerol) was added to 50 μg of σ⁷⁰ protein. 240units of cAMP-dependent kinase-catalytic subunit (Promega) was added,and the total volume was brought up to 99 μl with deionized water. Onemicroliter of [γ-³²P]ATP (0.15 mCi/μl) was added. The mixture wasincubated at room temperature for 30 minutes. The reaction mixture wasthen loaded onto a Biospin-P6 column (Bio-Rad) pre-equilibrated with1×kinase buffer and spun at 1,100×g for 4 minutes. The flow-through wascollected and stored at −20° C.

[0177] Probing

[0178] The blocked nitrocellulose was incubated in 10 ml of HYB bufferwith 4×10⁵ cpm/ml ³²P-labeled σ⁷⁰ for 3 hours at room temperature. Theblot was washed three times with 10 ml of HYB buffer for 3 minutes each.The blot was then dried and exposed to film or PhosphorImager (MolecularDynamics).

[0179] Co-immobilization

[0180] One milligram of His₆-tagged, truncated β′ was solubilized in 1ml of buffer C (20 mM Tris-HCl, pH 7.9, 200 mM NaCl, 5 mM imidazole, 0.1% (v/v) Tween 20, and 10% (v/v) glycerol) +8 M urea. Twenty microgramsof the protein solution were loaded onto 150 μl of Ni²⁺-NTA resin. Thecolumn was washed with 15 column volumes of buffer C +8 M urea, followedby a 15-column volume wash with buffer C to allow refolding. Then 30 μgof native σ⁷⁰ were loaded onto the column. The column was washed with 20volumes of buffer C. The bound proteins were eluted with 300 μl ofbuffer C+250 mM imidazole. Samples from the σ⁷⁰ flow-through, wash, andelution fractions were analyzed by SDS-PAGE.

[0181] Results

[0182] σ⁷⁰ Interacts Strongly with β′ Subunit and Weakly with β Subunitin Far-Western

[0183] Blot Analysis

[0184] Far-Western assays of dot blots were used to assess the bindingof σ⁷⁰ to individual a and A subunits outside of the core complex.Inclusion body proteins of β and β′ were separately solubilized in ureaand spotted onto nitrocellulose. Bovine serum albumin (BSA) was spottedas a control for nonspecific binding. The nitrocellulose was blocked andthe denaturant washed away. The blot was then probed with ³²P-labeledσ⁷⁰. Both β and β′ subunits bound σ⁷⁰, the BSA control did not.Identical dot blots were probed with control solutions lacking eitherthe kinase or 370 to ensure that the signal was not due to nucleotidebinding or phosphorylation of the β or β′ subunits. Neither control blotproduced a detectable signal. Thus, both β and β′ subunits canindividually bind σ⁷⁰.

[0185] σ⁷⁰ Interaction Specific for β/β′ in Far-Western Analysis

[0186] An additional test was performed to assess the specificity of thefar-Western analysis using σ⁷⁰ as a probe. A cell lysate from a logphase culture was separated by SDS-PAGE, blotted onto nitrocellulose,and probed with σ⁷⁰. The only strong signal produced had the samemobility as β and β′. The absence of other strong signals indicates thatσ⁷⁰ is not binding nonspecifically to β and/or β′. Minor bands wereobserved as expected, since there are other proteins that have beenshown to interact with σ⁷⁰ (activators, anti-σ, etc.) (Ishihama, 1993;Jishage et al., 1998).

[0187] A Strong, Specific Binding Site for G70 Is Located in the NTerminus of the β′ Subunit

[0188] To map the σ⁷⁰ interaction sites on β and β′, far-Westernanalysis of chemical cleavage products of the two large subunits wasperformed. The amino acid sequences of both were analyzed usingMacVector software (Oxford Molecular Group) to identify specificchemical cleavage sites. Based on this analysis, cleavage reagents werechosen that produced an array of products following partial digestionthat provide the highest resolution for mapping. Both N- and C-terminalHis₆-tagged constructs of β and β′ were subjected to cleavage underdenaturing conditions. The products of the cleavage reaction werepurified under denaturing conditions using Ni²⁺-NTA resin to isolatecleavage fragments containing a His₆ tag. These purified fragments werethen identified based on their mobility in SDS-PAGE, and their exactsize was determined based on the cleavage site which produced them. Whenthe cleavage fragments were fractionated by SDS-PAGE, they produced aladder of descending sized fragments with a common end (either N or Cterminus depending on the placement of the His₆ tag). The use of both N-and C-terminally His₆-tagged fragments allows the positiveidentification of both the N and C termini of the interaction domain.The σ⁷⁰ probe will only bind the fragments that have an intactinteraction domain. The N-terminally His₆-tagged β′ ladders produced byhydroxylamine and NTCB cleavage both contained several fragments thatretained the ability to bind σ⁷⁰. Thus, a large portion of the Cterminus of β′ can be removed without affecting σ⁷⁰ binding. Thesmallest fragment to bind σ⁷⁰ was the 1-309 amino acid fragment of β′ inthe hydroxylamine ladder. In the C-terminally His₆-tagged ladders, onlyfull-length β′ bound (σ⁷⁰. These results indicated that a strongspecific binding site is located within amino acids 1-309 of β′(β′₁₋₃₀₉). The β fragment ladders failed to produce signals strongenough to effectively map the interaction domain.

[0189] The resolution of chemical cleavage mapping was relatively lowdue to the limited number of cleavage sites on β′ for the reagentsavailable. To increase the number of proteolytic fragments that could beused in mapping, we used enzymatic cleavage. Specificity of cleavage bymany proteases is not as limited as with chemical cleavage reagents.Therefore, there are many more sites of cleavage and more fragments areproduced. Partial digests of N- and C-terminally His₆-tagged β′ wereconducted using trypsin and thermolysin. The fragments were againpurified, blotted, and probed with σ⁷⁰. However, even with the increasednumber of fragments, the interaction domain could not be narrowed fromits previous length of 1-309 amino acids.

[0190] Interaction Domain Narrowed to 60-309 aa by Far-Western Blottingwith Truncated Fragments

[0191] In trying to define this binding site more precisely, varioustruncated fragments were made using PCR. Using the β′₁₋₃₀₉ fragment as astarting point, constructs were made that were truncated at either the Nor C terminus. DNA coding for the truncated fragments was cloned intooverexpression plasmids. When cells containing these plasmids had beengrown to an A₆₀₀ of 0.6, expression was induced. The cells were onlyallowed to grow for 30 minutes after induction. A whole cell lysate fromeach culture was made and used for far-Western blotting assays. Shortexpression times kept the expression level of the induced proteincomparable with the other proteins in the lysate. The use of the wholecell lysate in far-Western blotting assays was an internal control toensure binding was specific for the protein of interest. This also meantthat the various proteins would not have to be purified and could beexpressed without purification tags. When constructs were made where theC terminus of β′₁₋₃₀₉ was truncated beyond amino acid 300, the bindingof (⁷⁰ was lost. However, the N terminus of the same fragment could betruncated up to 60 aa without diminishing the signal. β′₁₀₀₋₃₀₉ stillshowed binding, but at a lower level, and β′₁₅₀₋₃₀₉ did not bind σ⁷⁰.These results narrowed the σ⁷⁰ binding site to β′₆₀₋₃₀₉-Western blotexperiments using anti-β′ monoclonal antibodies were done to ensure thatthe protein fragments were being transferred to the nitrocellulose andthat they were fragments of β′.

[0192] Co-immobilization Assays Further Narrow Interaction Site toResidues 260-309 of β′

[0193] Ni²⁺-NTA co-immobilization assays were used to confirm and extendthe results that had been produced using far-Western blotting. Theproteins to be assayed for binding σ⁷⁰ were fused to His₆ purificationtags and overexpressed in the form of inclusion bodies. The inclusionbody protein was solubilized with 8 M urea and loaded onto Ni²⁺-NTAresin. The denaturant was washed away allowing the proteins to refoldwhile still remaining bound to the resin. Native σ⁷⁰ was then loadedonto the column. The column was washed, and the bound proteins were theneluted with imidazole. Any truncated protein that contained theinteraction domain for σ⁷⁰ would cause σ⁷⁰ to be bound and to be in theeluted fraction. The results of these binding experiments are consistentwith the far-Western blotting experiments in respect to defining theC-terminal boundary of the domain. β′₁₋₃₀₉ bound σ⁷⁰, while β′₁₋₃₀₀ andβ′₁₋₂₈₀ did not bind (70 Refolded β′₁₋₃₀₉ without a His₆ tag was mixedwith (70 and passed over the Ni²⁺-NTA to ensure the complex was notnonspecifically binding to the column. The complex passed through thecolumn and was not seen in the eluted fraction. As a control, BSA wasloaded onto a column containing β′₁₋₃₀₉. BSA was seen only in theflow-through and not in the eluted fraction, suggesting β′₁₋₃₀₉ bindsσ⁷⁰ specifically.

[0194] For the N-terminal boundary, the results showed that more of theN terminus could be removed without affecting σ⁷⁰ binding than was seenby the far-Western assay. Several N-terminally truncated fragments, allhaving aa 309 as the C-terminal boundary followed by a His₆ tag, wereconstructed and used in co-immobilization assays. Truncations toresidues 33, 60, 100, 178, and 200 still produced fragments capable ofbinding σ⁷⁰. β′₂₆₀₋₃₀₉, that was prepared and could be manipulatedefficiently, retained the ability to bind σ⁷⁰. To find the N terminus ofthe interaction domain, truncations greater than residue 240 were madefrom full-length β′. A truncation of the first 260 residues of β′(β′_(260-C)) bound σ⁷⁰, while β′_(270 -C) showed diminished binding, andβ′_(280-C) showed no detectable binding of σ⁷⁰. Taken together theseresults indicate that a strong σ⁷⁰ binding site on the core polymeraseis located within the residues 260-309 of β′.

[0195] Discussion

[0196] To date, several biochemical and genetic studies have contributedto what is known about the putative core binding domains on a, however,much less is known about the sites on core that bind σ (Gross et al.,1996). In the holoenzyme assembly pathway, β′ is added to the α₂βcomplex and then σ is added to form the holoenzyme (Ishihama, 1981).This would suggest that either the major a binding site is located on β′or is formed in cooperation with a and/or β upon β′ assembly into thecore enzyme. The isolation of σ⁷⁰.β′ complexes provides evidence for theformer (Luo et al., 1996). The results described hereinabove havelocalized a strong binding site for σ⁷⁰ on β′, as well as identified lowlevel binding affinity for σ⁷⁰ to β. Thus, β′ provides the major bindinginteraction for σ⁷⁰ in the holoenzyme while β adds a secondary bindinginteraction. Multiple core binding sites on a have been suggested inlight of σ mutations apparently affecting core binding that map outsideof conserved region 2.1 (Joo et al., 1997; Zhou et al., 1992; Joo etal., 1998; Sharp et al., 1999).

[0197] A strong binding site for σ⁷⁰ is located within residues 260-309of β′. A deletion of residues 201-477 of β′ has been reported previouslyto produce a mutant protein that could still form core but notholoenzyme (Luo et al., 1996). The problem with such deletion studies isthat one cannot conclude that the binding site is located in the regiondeleted, but merely that the region, when deleted, prevents correctformation of the interaction domain. Results obtained fromprotein-protein footprinting experiments indicated that a similar regionof β′ (residues 228-461) was physically close to σ⁷⁰ (Owens et al.,1998). There is difficulty in interpreting these results, since theassay gives indications of physical proximity of the proteins that donot necessarily correspond to protein-protein binding. From the findingsdescribed herein it can be concluded that a major σ⁷⁰ binding site islocated within these regions.

[0198] The σ⁷⁰ interaction domain on β′ contains several residueslocated in conserved region B (Jokerst et al., 1989). This region doesnot have any known function. Secondary structural predictions derivedfrom the PHD program (Rost et al., 1994) for residues 260-309 indicatesone helix from residue 264 to residue 283 connected by a loop to asecond helix from residue 292 to residue 309. These predicted helicesare also predicted to form coiled coils (Lupas et al., 1991). This is ofparticular interest, since similar predictions were made for residues355-391 of σ⁷⁰. These residues overlap conserved region 2.1. The crystalstructure of the protease-resistant fragment of σ⁷⁰ confirmed theprediction that the helix containing region 2.1 is forming a coiled coilwith conserved region 1.2 (Malhotra et al., 1996). Since coiled coilshave been shown to be involved in many protein-protein interactions(Landschulz et al., 1988; Gentz et al., 1989; O'Shea et al., 1989), thiswould suggest that α′₂₆₀₋₃₀₉ may be interacting with region 2.1 of σ⁷⁰.

[0199] Ordered fragment ladder far-Western blotting was used to map theOσ⁷⁰. binding site on β′ to within β′₆₀₋₃₀₉. This method relies on thefact that after the removal of the denaturant some fraction of theblotted protein will be able to refold and produce the properconformation for binding of the probe. The specificity of the assay wasdemonstrated by probing whole cell lysates and identifying β′ as themajor binding interaction. The combination of specific chemical cleavageof proteins and far-Western blotting provided a very rapid and effectiveway to localize this protein-protein interaction. Cloning and screeningindividual, truncated fragments was necessary only after the interactiondomain had been targeted. Having to make truncations of β′ all along itslength would have been a long and tedious process. The protein cleavageand Ni²+column purification procedures can be done in one day, thusmaking the assay more expedient and less tedious.

[0200] To confirm and extend the results obtained with far-Westernblotting, Ni²⁺ co-immobilization assays were performed. Theseexperiments also demonstrated that fragments from the N terminus toresidue 309 could still bind σ⁷⁰, while removal of just 9 C-terminalresidues to aa 300 would abolish binding. The results obtained from theN-terminally truncated fragments in these assays gave much betterresolution of the binding site location than was obtained fromfar-Western assays. Up to 260 residues could be removed from the Nterminus without affecting σ⁷⁰ binding. When 270 residues were removed,binding of σ⁷⁰ was diminished but not abolished, suggesting that eitherpart of the binding site had been removed or the binding site was intactbut hindered from refolding due to the loss of upstream residues. Toensure that the binding site was what was actually mapped and not just aregion required for proper folding of the actual binding site, proteinfragments were made from 260-309 of β′ and shown to be sufficient forbinding. The difference in the identified interaction domain sizebetween the far-Western assay (β′₆₀₋₃₀₉) and the co-immobilization assay(β′₂₆₀₋₃₀₉) is consistent with the properties of each assay. Thefar-Western assay requires the interaction domain to refold and properlypresent the binding site while some portion of the protein is attachedto the nitrocellulose membrane. As such the proteins are moreconformationally restricted than proteins bound only at one terminus asin the Ni²⁺-NTA co-immobilization assay. Therefore, more of the proteinlength is required to form a scaffold-like structure to keep theinteraction domain away from the membrane surface. The combination ofmapping methods provides a rapid, high resolution procedure foridentification of protein interaction domains.

EXAMPLE 3 Mutational Analysis of β′₂₆₀₋₃₀₉

[0201] Materials and Methods

[0202] Construction of Plasmids

[0203] Plasmid characteristics are described in FIG. 7 and Tables 3-4.Plasmids pTA577 and 600-620 were made from the base plasmid pRL663 (Wanget al., 1995). Single HindIII and BamHI restriction sites were insertedinto the rpoC gene of pRL663 via silent mutagenesis to create pTA577.pTA561 was created in the same manner as pTA577 except pRL308(Weilbaecher et al., 1994) was the starting plasmid. The Hindfl andBamHI restriction sites were used to insert PCR generated DNA fragmentscontaining the various mutations to generate pTA600-609. For pTA620,containing a truncated rpoC fragment coding for β′ residues 1-319,pRL663 was cut with XbaI HindlU for insertion of the PCR generated rpoCtruncation. The σ⁷⁰ binding site was mapped to 260-309 of β′, however,some of the constructs were engineered to extend to residue 319. Thiswas done to incorporate the BamHI site mentioned above. Thus, thevarious mutations were moved into the new plasmid to create pTA610-619.There was no observed difference in the properties of the fragmentsending to residue 309 as opposed to those ending to residue 319.

[0204] Plasmids pTA145, 655, 658, 660 and 661 were constructed byinserting PCR generated rpoC fragments, coding for β′₂₄₀₋₃₀₉ wild typeor the various mutants, into the NdeI-XhoI restriction sites of pET24a.C-terminal His₆ tags were incorporated in the reverse primers for theseinserts to fuse the purification tags to the expressed proteins. TABLE 3Plasmid β′ residues Mutation Modifications Reference pRL308 1-1407 nonenone Weilbaecher et al., 1994 pRL663 1-1407 none C-terminal His₆ Wang etal., 1995 pTA561 1-1407 silent none This work pTA577 1-1407 silentC-terminal His₆ This work pTA600 1-1407 N266D C-terminal His₆ This workpTA601 1-1407 Y269A C-terminal His₆ This work pTA602 1-1407 R275QC-terminal His₆ This work pTA603 1-1407 K280E C-terminal His₆ This workpTA604 1-1407 R293Q C-terminal His₆ This work pTA605 1-1407 E295KC-terminal His₆ This work pTA606 1-1407 R297S C-terminal His₆ This workpTA607 1-1407 Q300E C-terminal His₆ This work pTA608 1-1407 A302DC-terminal His₆ This work pTA609 1-1407 N309D C-terminal His₆ This workpTA610 1-319 N266D none This work pTA611 1-319 Y269A none This workpTA612 1-319 R275Q none This work pTA613 1-319 K280E none This workpTA614 1-319 R293Q none This work pTA615 1-319 E295K none This workpTA616 1-319 R297S none This work pTA617 1-319 Q300E none This workpTA618 1-319 A302D none This work pTA619 1-319 N309D none This workpTA620 1-319 silent none This work

[0205] TABLE 4 Far- Assembly Assembly Plasmid Western Growth Eσ70 Eσ32 #binding at 42° C. holo holo Toxic   wt + + + + + R297L +/− − + L299D − −− E301K + + + E301P − − − R270L + + V272D − − N274D + + N274P − − 600N266D + − +/− − + 601 Y269A + + + +/− − 602 R275Q − − − − − 603K280E + + + + + 604 R293Q +/− +/− +/− + + 605 E295K − − − − − 606 R297S− +/− − + + 607 Q300E + + + +/− + 608 A302D − − − − − 609 N309D + + + ++

[0206] Expression and Purification of 370

[0207] The cells were grown to an A₆₀₀ between 0.6-08 in 1 L cultures at37° C. in LB medium with 100 μg/ml ampicillin. Isopropylβ-D-thiogalactoside (IPTG) was then added to a concentration of 1 mM.Three hours after induction, the cells were harvested by centrifugationat 8,000×g for 15 minutes and frozen at −20° C.

[0208] The cell pellet from a 1 L culture was thawed and resuspended in10 ml of lysis buffer (40 mM Tris-HCl, pH 7.9, 0.3 M KC1, 10 mM EDTA and0.1 mM phenylmethylsulfonyl fluoride) and lysozyme was added 0.1 mg/ml.The cells were incubated on ice for 15 minutes then sonicated 3×60second bursts. The recombinant protein in the form of inclusion bodieswas separated from the soluble lysate by centrifugation at 27,000×g for15 minutes. The inclusion body pellet was resuspended by sonication in10 ml of lysis buffer +2% (w/v) sodium deoxycholate (DOC). The mixturewas centrifuged at 27,000×g for 15 minutes and the supernatant wasdiscarded. The DOC-washed inclusion bodies were resuspended in 10 mldeionized water and centrifuged at 27,000×g for 15 minutes. The waterwash was repeated and the inclusion bodies were aliquoted into 1 mgpellets and frozen at −20° C. until use.

[0209] σ⁷⁰ inclusion bodies (10 mg) were solubilized, refolded andpurified according to a variation of the procedure of Gribskov andBurgess (1986). The inclusion bodies were solubilized by resuspension in10 ml of 6 M guanidine-HCl. The proteins were allowed to refold bydiluting the denaturant 64-fold with buffer A (50 mM Tris-HCl, pH 7.9,0.5 mM EDTA, and 5% (v/v) glycerol) in 2-fold steps over 2 hours. Onegram of resin (DEAE-cellulose, Whatman) was added and mixed with slowstirring for 24 hours at 4° C. The resin was then collected in a 10 mlcolumn, washed, and the protein eluted with a gradient from 0.1 to 1.0 MNaCl in buffer A. The Uσ⁷⁰ fractions were pooled and dialyzed overnightagainst 1 L of storage buffer (50 mM Tris-HCl, pH 7.9, 0.5 mM EDTA, 0.1M NaCl, 0.1 mM DTT and 50% (v/v) glycerol) and stored at −20° C.

[0210] Quantitative Western Blotting

[0211] Protein samples to be quantitated were subjected toSDS-polyacrylamide gel electrophoresis (PAGE). The proteins wereelectrophoretically transferred out of the gel onto 0.05 μmnitrocellulose. The blot was blocked in Blotto and probed withmonoclonal antibodies (MAbs). The signal was generated using theECL+system (Amersham) and detected on a Storm Fluoroinager (MolecularDynamics). The signal was quantitated using ImageQuant software(Molecular Dynamics).

[0212] Far-Western Blotting

[0213] Cells containing truncated β′ expression plasmids pTA610-620 weregrown to an A₆₀₀ of 0.6-0.8 and induced with 1 mM IPTG. The cells weregrown for an additional 30 minutes. A 200 Fll sample was removed andsonicated 3×30 seconds. Twenty μl of glycerol and 20 Il of SDS-samplebuffer were added and heated for 2 minutes at 95° C. then stored at −20°C. until use. The lysates were separated by SDS-PAGE. The proteins wereelectrophoretically transferred onto 0.05 μm nitrocellulose. Thenitrocellulose was blocked by incubating in HYB buffer (20 mM Hepes, pH7.2,200 mM KCl, 2 mM MgCl₂, 0.1 μM ZnCl₂, 1 mM DTT, 0.5% (v/v) Tween 20,1% (w/v) non-fat dry milk) for 16 hours at 4° C.

[0214] Labeling of σ⁷⁰ was done in a 100 μl of 2×kinase buffer (40 mMTris-HCl, pH 7.4, 200 mM NaCl, 24 mM MgCl₂, 2 mM DTT) was added to 50 μgof HMK-σ₇₀ protein. Two hundred and forty (240) U of cAMP-dependentkinase catalytic subunit (Promega) was added and the total volume wasbrought to 99 μl with deionized water. One microliter of γ-³²P-ATP (0.15mCi/ml) was added. The mixture was incubated at room temperature for 30minutes. The reaction mixture was then loaded onto a Biospin-P6 column(BioRad) pre-equilibrated with 1×kinase buffer and spun at 1100×g for 4minutes. The flow-through was collected and stored at −20° C.

[0215] The blocked nitrocellulose was incubated in 10 ml of HYB bufferwith 4×10⁵ cpm/ml³²P-labeled σ⁷⁰ for 3 hours at room temperature. Theblot was washed three times with 10 ml of HYB buffer for 3 minutes each.The blot was dried and the signal was visualized with a PhosphorImagerand quantitated with IMAGEQUANT software (Molecular Dynamics).

[0216] Growth Assessment

[0217] Plasmids pTA577, 600-609 (0.1 μg) were transformed into strainRL602 (Weilbaecher et al., 1994; Ridley et al., 1982). After heat shockand incubation on ice, 300 μl of LB was added to the 50 μl cell mixture.Ten μl of the transformation reaction was spotted onto LB plates plusampicillin (100 μg/ml) and incubated at 30° C. Another 10 μL was spottedonto plates and incubated at 42° C. The plates were incubated between24-48 hours and assessed for growth.

[0218] Purification of Core/holoenzyme Complexes

[0219] One L flasks containing 200 ml of LB with ampicillin (100 μg/ml)and IPTG i(0.15 mM) were inoculated with 200 μl from an overnightculture of cells containing plasmids pTA561, 577, 600-609. The cultureswere grown at 37° C. with shaking until the A₆₀₀=0.4 for log phaseassays and 2 hours longer (A₆₀₀ about 2.0) for the early stationaryphase assays. The cells were harvested by centrifugation at 6,000 rpmfor 10 minutes and stored at −20° C. until use. The cell pellets wereresuspended in 5 ml TE (10 mM Tris-HCl, pH 7.9 and 0.1 mM EDTA) plus0.15 M NaCl and lysozyme (0.1 mg/ml) then incubated on ice for 15minutes. The cells were sonicated 2×30 seconds and centrifuged for 25minutes at 27,000×g to pellet the insoluble material. The supernatantwas loaded onto a 1.5 ml immunoaffinity column containing thepolyol-responsive, anti-β′ monoclonal antibody (MAb), NT73 (Thompson etal., 1992). The column was washed with 15 ml TE plus 0.15 M NaClfollowed by a second wash with 10 ml TE plus 0.5 M NaCl. The protein waseluted from the column with 4 ml TE plus 0.7 M NaCl and 30% propyleneglycol. The eluted sample (4 ml) was diluted with 6 ml buffer B (20 mMTris-HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole, 0.1% (v/v) Tween 20, and10% (v/v) glycerol) and loaded (2x) onto 500 ,ul of Ni²⁺-NTA resin. Theresin was washed (2×) with 5 ml of buffer B and eluted with 0.5 mlbuffer B plus 0.25 M imidazole. Samples from the elution fractions wereassayed by Western blot as described above using Mabs to each subunit ora factor. The secondary antibodies were horseradish peroxidase-labeledgoat antimouse IgG antibodies and the signal was generated using theECL+substrate system (Amersham) and detected using the STORMFluorolmager and quantitated with IMAGEQUANT software (MolecularDynamics).

[0220] Results

[0221] Mutational Design

[0222] The Coils prediction program (Lupas et al., 1991) scored both ofthe predicted α-helices of β′₂₆₀₋₃₀₉ as having a high probability offorming coiled coils (FIG. 3a). To test this prediction two β′ mutantswere constructed with proline residues inserted into either helix. Theseβ′ mutants were no longer predicted to form helices or coiled coils.When assayed for function in both the far-Western and in vivo growthassays, both mutants were found to be nonfunctional. This indicated thatthe helical/coiled coil structure in this region was important forfunction. However, the solubility of these mutant proteins was not 100%,so their loss of function could simply be due to gross folding defects.Further analyses were directed for the most part on the “e” and “g”positions of the helices. The e and g residues of coiled coils oftenengage in interhelical interactions such as the formation of ionicinteractions or salt bridges (Cohen et al., 1986; Chao et al., 1998).Such interactions in this case could be intramolecular (between the twohelices of °′₂₆₀₋₃₀₉) forming a coiled coil structure necessary forbinding by the a subunit (FIG. 3b). Alternatively, the e and g residuesof β′₂₆₀₋₃₀₉ could be making intermolecular contacts with helices of aupon binding. Change-of-charge mutations were prepared at these residuesof β′ and the effect of the mutation on binding determined (FIG. 3b).Two of the mutations described do not involve e or g residues. Based onthe findings that tyrosine and arginine residues are often located in“hot spots” of protein-protein interactions (Bogan et al., 1998), thetyrosine residue at position 269 was changed to an alanine and thearginine residue at position 297 to a serine. It had been determinedthat insertion of a leucine at position 297 generated a β′ subunit thatwas nonfunctional for binding σ⁷⁰ (data not shown). Therefore, it was ofinterest to determine if a less drastic mutation at this position wouldalso affect ay binding. Several of the mutations in the β′₂₆₀₋₃₁₉ regiondisrupt interaction with σ⁷⁰ in a far-Western assay.

[0223] Far-Western blotting had been used to map a σ₇₀ binding site tothe N-terminal region of the β′ subunit (Example 2). This method wasinitially employed to detect the functionality of the β′ mutants. Themutations were cloned into a gene fragment coding for amino acidresidues 1-319 of the β′ subunit. Cells containing these genes wereinduced for a short period to give moderate levels of the β′ fragment,comparable to other proteins in the extract. Samples were analyzed forbinding σ⁷⁰ by far Western analysis The amount of (σ⁷⁰ probe bound byeach β′₁₋₃₁₉ mutant fragment was compared to the amount bound by wtβ′₁₋₃₁₉ fragment. Each signal was normalized to the amount of β′₁₋₃₁₉contained in the supernatant as determined by Western blotting.

[0224] Five of the mutations (R275Q, R293Q, E295K, R297S, and A302D)were greatly reduced in their ability to bind σ⁷⁰ (FIG. 4). The Q300Eand N309D mutations had the opposite effect, binding more σ⁷⁰ than wildtype β′₁₋₃₁₉Q300E exhibited an increase in relative binding of greaterthan 7-fold. There were no effects on binding seen with the N266D,Y269A, or K280E mutations.

[0225] Growth with Mutant β′

[0226] To assess the importance of the σ⁷⁰ binding site in vivo, theability of mutant β′ subunits to function as the cell's sole source ofβ′ was assessed. Plasmids containing either mutant or wild type, fulllength β′ were transformed into strain RL602 (Weilbaecher et al., 1994;Ridley et al., 1982). The chromosomal rpoC gene of RL602 has an ambermutation that prevents functional β′ from being produced in the absenceof a suppressor tRNA. RL602 also has a chromosomal,temperature-sensitive, amber suppressor. At the permissive temperature(30° C.) the amber suppressor is active and allows chromosomal β′ to beproduced and the cell can grow. The amber suppressor is not active atthe non-permissive temperature (42° C.). Therefore, at 42° C.,chromosomal β′ is not made and the cell cannot grow without anothersource of β′. If the plasmid-derived β′ can complement the loss of β′,the cells will grow and form colonies on plates at the non-permissivetemperature. If the mutant β′ cannot complement, there will be no growthon the plates at this temperature.

[0227] Three of the mutations that were defective for ac binding in thefar-Western assay (R275Q, E295K, and A302D) could not support growth atthe non-permissive temperature, indicating that these mutations werealso defective in binding σ in vivo (FIG. 5). N266D, a mutation that hadno detectable effect in the far-Western assay, allowed some growth atthe non-permissive temperature but not enough to be considered wildtype. In contrast, the R293Q and R297S mutations that did not bind σ⁷⁰in the far-Western assay could support growth in vivo. Other mutations(Y269A, K280E, Q300E, and N309D) had no detectable effects on growth.Expression levels for nonfunctional β′ mutants were determined to beequivalent to that of plasmid-derived, wild type β′ when grown at 37° C.(data not shown).

[0228] Core/Holo Assembly

[0229] To evaluate the potential assembly defects caused by the variousmutations, His₆-tagged, mutant β′ subunits were expressed in cells thatwere also expressing wild type, chromosomal β′ proteins. A Ni²⁺-NTAcolumn was used to purify the mutant β′ subunits together withassociated cell proteins. An immunoaffinity column was used to clean-upthe samples in order to reduce any non-specific binding to the Ni²⁺-NTAcolumn.

[0230] All of the mutant β′subunits tested retained the ability toassemble into the core enzyme demonstrated by the association of the aand P subunits throughout the purification (FIGS. 6a and 6 b). Again,mutations R275Q, E295K, and A302D caused defects in binding σ⁷⁰ in bothlog and stationary phase samples. Also reduced in Eσ⁷⁰ formation wereN266D in both log and stationary phase samples and R297S in log phasesamples. Q300E again showed properties of binding σ⁷⁰ better than wildtype. Y269A, K280E, R293Q, and N309D had no detectable affect on Eσ⁷⁰assembly. When a non-His₆ tagged β′ was expressed from the plasmid,there was no detectable nonspecific binding to the Ni²⁺-NTA column.

[0231] All of the sample eluates were also assayed for the presence ofany minor a species. The only minor σ's whose concentrations weresufficient for detection were σ³² in log phase and σ³² and (F instationary phase samples. The results for these σ's were essentially thesame as for σ⁷⁰ with the exception of mutants R297S and Q300E. Instationary phase samples from the Q300E mutant the σ³² and σ^(F) levelsare greatly reduced while the Yσ⁷⁰ levels are above wild type. The logphase samples for this mutant also contained a decreased amount of σ³²,indicating a defect in Eσ³² formation but not as severe as in stationaryphase.

[0232] Molecular ModelingT

[0233] Recently, Zhang et al. (1999) published the crystal structure ofT. aquaticus RNA polymerase. The β′₂₆₀₋₃₀₉ region of E. coli RNApolymerase has a high degree of sequence conservation with its T.aquaticus homolog (FIG. 8a). This region of the T. aquaticus a subunitforms a “coiled coil-like” structure. When the mutations studied hereinare modeled onto the T. aquaticus structure using the Rasmol softwareprogram (Sayle et al., 1998), those that are most defective in a bindingare grouped on one face of the coiled coil. Those that had defectivephenotypes in some assays but not others are on the outer edges of thisface. Mutations that had no detectable effects are clustered on theopposite face of the coiled coil with the exception of N309D which islocated at the very C terminus of the coiled coil immediately next tothe “rudder” (FIGS. 8b and 8 c).

[0234] Discussion

[0235] Binding of various a factors to the core polymerase is a majorstep in the process of global gene expression and regulation. It is notknown if this step is part of the regulation, via a competition forbinding to a limited core population, or merely a straight-forwardbinding of free σ's to an excess of core. If there is competition amongpopulations of a species for core binding, that competition may beinfluenced by binding specificity of the σ's. In light of the highsequence conservation of most σ species, it has been hypothesized thatall σ factors bind to the same locations on the core enzyme (Helmann etal., 1988). As described herein, the in vivo binding site on β′ of RNApolymerase for σ⁷⁰ was identified. Moreover, this binding site is alsoinvolved in binding at least some of the minor σ factors. Further, themethods, compositions and compounds described herein are useful toidentify important residues in core RNA polymerase for a binding, and todefine a potential binding interface for the σ-core interaction.

[0236] The mutational analysis approach, which was designed to look forloss of σ⁷⁰ binding by targeting residues of the 260-309 region of theβ′ subunit that were identified as occupying e or g positions in thepredicted coiled coil structure, resulted in three classes of mutations:nonfunctional for a binding in all assays tested; nonfunctional in someassays but not others; and those that were functional in all assaystested. The first group contains mutations R275Q, E295K, A302D. Thesethree mutations were nonfunctional for σ⁷⁰ binding in vitro and in vivo,indicating that they play a very important role in binding σ⁷⁰ Arginine275 is located near the C terminus of the first of the two putativehelices while glutamate 295 and alanine 302 are in the middle and nearthe C terminus of the second helix, respectively. This confirms thatboth predicted helices of β′₂₆₀₋₃₀₉ were involved in binding σ⁷⁰ . Themutations made at these residues were the only ones tested that couldnot support any detectable growth when the expression of the chromosomalβ′ subunit was turned off. This is significant in light of the fact thatthese mutant β′ subunits, along with all those tested in this study, hadno detectable defect in interactions with the a and p subunits necessaryto form the core enzyme. Thus, there are no gross folding defects thatare responsible for the lack of σ⁷⁰ binding.

[0237] It is possible that the local structure of these mutant proteinsis disturbed. Sequence analysis of all 10 mutant subunits predicted nochange in secondary structures as compared to the wild type protein(Rost et al., 1994; Munoz et al., 1994). The A302D change would be themost likely, though, of the three group 1 mutations to be disturbing thelocal structure. This introduces a bulky charged side chain in place ofa single methyl group. Also, based on the crystal structure of the T.aquaticus core RNA polymerase (Zhang et al., 1999), the A302 a carbon isdirected more toward the opposite helix of the coiled coil than are theside chains of R275 or E295 which are solvent exposed. If R275Q andE295K are not affecting the local β′ structure then most likely thenegative σ⁷⁰ binding properties are coming from stearic hindrance,charge repulsion, or loss of a specific interaction with the σ subunit.

[0238] The group 2 mutants, N266D, R293Q, and R297S, are of particularinterest since they seem to have some function depending on the assay inwhich they are analyzed. R293Q and R297S were not functional for invitro σ⁷⁰ binding in far Western assays but could support growth andwere able to form core enzymes that were capable of binding σ⁷⁰,although R297S does cause a decrease in the binding efficiency of themutant core enzyme in log phase. The differences in the in vivo and invitro assay results for these mutants can be explained in multiple ways.First, a positive result from the far Western assay requires that a β′fragment (1-319) refold the secondary structure needed to bind σ⁷⁰ whilepart of the protein is immobilized on a membrane. Therefore, mutationsfound to cause defects may be introducing in vitro folding deficiencies.Secondly, the in vivo assays are analyzing a binding to the multisubunitcore enzyme and not just an individual subunit or fragment. A great dealof evidence has been reported suggesting multiple binding sites on coreRNA polymerase for the a factor (Sharp et al., 1999: Joo et al., 1998;Nagai et al., 1997; Owens et al., 1998). Thus, loss of one of thosesites may be compensated for by the remaining binding interactions.While R293Q and R297S mutations are disrupting a binding toβ′_(260-309,) they are not obstructing σ⁷⁰ from making its othercontacts on core polymerase.

[0239] In contrast to the previous group 2 mutations, N266D had noeffect on σ⁷⁰ binding to β′, but caused reductions in Eσ⁷⁰ formationcomparable to group 1 mutations and had a weak growth deficiency. N266is located at the base of the coiled coil and when mutated could changethe local structure. This change may be causing a shift in theorientation of the coiled coil with respect to the rest of the coreenzyme. This would not affect the binding of σ⁷⁰ to the coiled coil butmay disrupt other contacts normally made by σ⁷⁰ with core.

[0240] The group 3 mutants, Y269A, K280E, Q300E, and N309D, were allfully functional indicating that these residues are not making criticalcontacts with σ⁷⁰. The Q300E change was rather interesting. Thismutation seems to cause an increase in binding of σ⁷⁰ to β′. The largeincrease in relative binding seen in the far Western was not as dramaticin vivo possibly due to the σ⁷⁰-core interaction having a larger Keqthan the σ⁷⁰-β′ interaction. Although, the assembly of σ⁷⁰ for thismutant still was almost twice that of wild type. Inhibitors based oncoiled coil interactions have proven to be useful in disrupting suchprocesses as viral entry into cells and topoisomerase activity (Eckertet al., 1999; Wild et al., 1994; Frere-Gallois et al., 1997). Inhibitorsof the σ-core interaction may be useful as antibacterial therapeutics.In particular, the Q300E mutation may provide useful information onincreasing the binding constant of such an inhibitor.

[0241] The alternative σ factors have been thought to bind to the samesites on core RNA polymerase as σ⁷⁰. Mutating conserved residues ofdifferent a species will disrupt core binding (Sharp et al., 1999).Traviglia et al. (1999) used tethered Fe-EDTA cleavage to determine thatseveral of the minor a species of E. coli are in close proximity to thesame regions of core RNA polymerase as σ⁷⁰ within the Eσ complex. Atleast for σ³² and σ^(F), minor a factors do bind one of the same siteson core as σ⁷⁰. Though they are binding to the same site, there is somedifference in the manner of binding. The Q300E mutation that increasedbinding of σ⁷⁰ had the opposite effect, especially in stationary phase,on σ³² and σ^(F). R297S also had different binding properties for the σfactors. This mutation caused an increased binding of the minor sigmasand reduced binding of σ⁷⁰. It is interesting that these mutations bothhad opposing effects on σ⁷⁰ and the minor σ's, although only two minorσ's were at detectable levels. This suggests that changes in the localenvironment could favor or hinder minor σ binding as a whole as comparedto σ⁷⁰.

[0242] Finally, the crystal structure of T. aquaticus core RNApolymerase has been of great utility in trying to understand the resultsof the mutations. Based on the computer predictions and the mutationalresults described herein, it could not have been concluded thatβ′₂₆₀₋₃₀₉ formed a coiled coil structure. However, combining thisinformation with the T. aquaticus versus E. coli β′ sequence alignmentand the T. aquaticus crystal structure, it is clear that β′₂₆₀₋₃₀₉adopts a coiled coil conformation. Upon σ binding though, it is notclear what structure this region takes on. Conserved region 2.1 σ⁷⁰,implicated in core binding (Lesley et al., 1989), forms a coiled coilwith region 1.2 in the crystal structure of the σ⁷⁰ protease-resistantdomain (Malhotra et al., 1996). Also, a predicted coiled coil in σ⁵⁴ ofE. coli has been found to be important in the σ-core interaction (Hsiehet al., 1999). These ay factor structures may be interacting withβ′₂₆₀₋₃₀₉ to form a four helix coiled coil. It is also known that the σfactor undergoes a conformational change upon binding core (Nagai etal., 1997; Callaci et al., 1998; McMahan et al., 1999). This may becaused by a rearrangement of the coiled coils to form new contacts (Grumet al., 1999; El-Kettani et al., 1996).

[0243] From the clustering of the group 1 mutations on the same face ofthe coiled coil structure while having the group 2 mutations on theedges of the cluster and the group 3 members on the opposite side of thecoiled coil, the binding interface for σ⁷⁰ on β′ has been defined.Recent work has localized the region of σ⁷⁰ that is interacting withβ′₂₆₀₋₃₀₉ to a peptide containing a portion of the nonconserved regionand region 2.1-2.2 of the σ⁷⁰ subunit (Burgess et al., 1998). β′ region198-237 was identified by Brodolin et al. (2000) as interacting with thenontemplate strand of the lacUV5 promoter which also is known to becontacted by region 2.4 of σ⁷⁰ (Siegele et al., 1989; Waldburger et al.,1990).

EXAMPLE 4

[0244] Luminescence Resonance Energy Transfer (LRET) to Monitor σ⁷⁰ toCore RNA Polymerase Interactions

[0245] The bacterial transcription machinery appears to offer anattractive target for drug discovery and drug design as it is highlyconserved among the bacterial kingdom, but significantly different fromeucaryotes. Any inhibitor of the assembly of a sigma factor with coreRNAP to form the holoenzyme would inhibit the initiation oftranscription in general and therefore prevent growth and eventuallysurvival of a cell. The postulated region mainly responsible for corebinding (region 2.1-2.2, FIG. 20) of bacterial transcription factors(σ⁷⁰ in E. coli) shows a remarkably high identity (>80%) and is alsohighly similar among the minor sigma factors of bacteria (Lesley andBurgess, 1989; Lonetto et al., 1998). Additionally, β′ (FIG. 20) in coreRNAP exhibits a very high sequence conservation in the sigma-bindingregion (between residues 260-309 in β′ of E. coli) (Arthur et al., 2000;Arthur and Burgess, 1998). These homologies suggest a highly conservedstructure within the holo form of RNAP, whose formation is crucial forcorrect initiation of transcription. Any inhibitor of this interactioncan thus be expected to be a broad spectrum antibiotic. No 67 homologhas been found in mammalian cells except for sigma factors inmitochondria (Tracy and Stern, 1995) and chloroplasts (Allison, 2000),however, they do not show a significant homology to their prokaryoticcounterparts. This fact implies that there is very little chance of apotential new antibiotic interfering with eukaryotic RNA polymeraseassembly, which could otherwise lead to serious side effects, if it wereused as a drug.

[0246] In order to screen for inhibitors of RNAP assembly with a, asimple, fast and reliable assay is needed for the formation of theσ⁷⁰-β′-complex. Electrophoretic mobility shift (EMS) assays andfar-Western blots were shown to be very helpful in identifying thebinding regions within E. coli RNAP (Burgess et al., 2000), but for apotential high-throughput screen a more rapid and preferably homogenousassay with a very sensitive signal-to-noise ratio would be desirable. Afluorescence-based probe assay was chosen to monitor complex formation.In this respect FRET (Fluorescence Resonance Energy Transfer) offers asystem that can create the desired signal upon complex formation(Selvin, 1995; Selvin, 2000; Stryer, 1978). FRET occurs when twoappropriate dyes get into proximity of less than about 75 A depending ontheir spectral properties. The energy transfer between these dyes istransmitted via dipole-dipole interactions. The acceptor is therebysensitized and can now fluoresce with its peculiar wavelength. Bothemissions have different wavelengths and can be monitored separately.This can give information about amount of and distance between the twodyes. A quantitative description of the effect is based on the Foirstertheory that describes the decrease of energy transfer as inverselyproportional to sixth-power of the distance between the two dyes. Hence,the energy transfer and thereby the distance between the dyes can bedetermined by measuring the intensity of the emissions and their decay.

[0247] LRET (Luminescence Resonance Energy Transfer) is a modifiedversion of this effect (Selvin, 1999). Different from FRET, the donor isan organic dye coupled to a lanthanide complex. This difference offersadvantageous spectroscopic features. A large stokes shift (distancebetween excitation and emission wavelength) avoids a strong cross talkbetween the instrumental excitation source and the emission. Narrowemission lines allow an accurate separation of donor and acceptorsignal. The lifetime of most lanthanides like Eu and Tb aresignificantly longer (milliseconds) than the vast majority of thecommonly used organic dyes like Cy5 (nanoseconds). Measuring intime-resolved fluorescence mode allows one to start signal acquisitionafter the background fluorescence and the intrinsic acceptorfluorescence have decayed. Thus, only the donor-sensitized emission ofthe acceptor can be measured which leads to a very good signal-to-noiseratio. The two dyes used in the assay are shown in FIG. 10.

[0248] Heyduk and co-workers have used LRET to measure DNA binding toσ⁷⁰ in holoenzyme using the same pair of dyes (Heyduk and Heyduk, 1999).For the assay described hereinbelow, an IC5-labeled β′-fragment(residues 100-309 N-terminally fused to a heart-muscle kinase (HMK)recognition site and a His₆-Tag) was substituted for the Cy5-labeledpolynucleotide. For the resulting homogenous assay, d⁷⁰ was labeled witha Europium-DTPA-AMCA complex as a donor and theHMK-His₆-β′(100-309)-fragment with the Cy5-analogue IC5-maleimide(Dojindo, Japan). FIG. 11 illustrates the assay. Upon complex formation,LRET occurs. Complex formation between 67 and β′ is monitored simply bylooking at the delayed emission of the acceptor as an opticallymeasurable signal of complex formation. The assay can be performed in amulti-well plate and measured by a multi-plate reader to accomplish ahigh-throughput of a large number of samples from any chemical libraryin an automated way. Typical reaction volumes are 10-200 μL where thecomponents including the test substances are mixed directly in themulti-well plate before the plate is measured in the reading device. Thevery sensitive nature of such a fluorescence-based assay (typically inthe low nanomolar range) provides good accuracy and signal-to-noiseratio, avoiding false positive hits in the measurement. It also permitsa low usage of labeled protein and substrates, which cuts down onoverall costs.

[0249] Methods

[0250] Buffers

[0251] The following buffers were used: NTGED-Buffer (50 mM NaCl, 50 mMTris-HCl, pH 7.9, 5% glycerol, 0.1 mM EDTA, 0.1 mM DDT); TGE-Buffer (50mM Tris-HCl, pH 7.9, 5% glycerol, 0.1 mM EDTA); NTG-buffer=FRET-Buffer(50 mM NaCl, 50 mM Tris-HCl, pH 7.9, 5% glycerol); TNTwGu-buffer (50 mMTris-HCl, pH 7.9, 500 mM NaCl, 0.1 v/v % Tween 20, 6 M Gu-HCl); NativeSample-buffer (200 mM Tris-HCl, pH 8.8, 20 v/v % glycerol, 0.005%bromphenol blue); and Storage-Buffer (50 mM NaCl, 50 mM Tris-HCl, pH7.9, 50% glycerol, 0.5 mM EDTA, 0.1 mM DDT).

[0252] Overproduction of HMK-His₆-β′,(100-309) and σ⁷⁰ (C132S, C291S.C295S S442C)

[0253] The plasmid pTA133 (Arthur and Burgess, 1998; FIG. 12) is aderivative of the expression vector pET28b(+). First a HMK-site wasinserted into the 5′-start of the MCS, then the partial β′ sequence wascloned into the resulting vector to yield a chimera (25 kDa) with theamino acid sequence MARRASVHHHHHHM -terminally fused to β′,(100-309).The HMK recognition site is underlined.

[0254] Plasmid pSigma7o(442C) (Heyduk and Heyduk, 1999; FIG. 13) isderived from the σ⁷⁰-expression system pGEMD (Igarashi and Ishihama,1991; Nakamura, 1980) that had a HindlII fragment containing the rpoDgene from E. coli cloned into a pGEMX-1 (Promega) vector. It is a T7expression system allowing controlled induction by IPTG and selectionwith ampicillin. The plasmids were transformed into BL21(DE3) (Novagen)for expression. The cells were grown in 1 L cultures at 37° C. in LBmedium with 100 μg/ml ampicillin. The cultures were grown to an OD600between 0.5-0.7 and then induced with 0.5 mM isopropylβ-D-thiogalactoside (IPTG). Two hours after induction, the cells wereharvested by centrifugation at 8,000×g for 15 minutes and frozen at −20°C. until use.

[0255] Purification of inclusion bodies

[0256] A cell pellet (1-2 g wet weight) was resuspended in 10 niLNTGED-buffer+10 mM EDTA and 100 gg/mi lysozyme. The cells were incubatedon ice for 30 minutes then sonicated three times in 60 second bursts at4° C. Triton X-100 (1% v/v) was added and vortexed. The recombinantprotein in the form of inclusion bodies was separated from the solublelysate by centrifugation at 25,000×g for 15 minutes. At each step, a 100liL sample was taken for SDS-PAGE, which is shown in FIG. 14. Theinclusion body pellet was resuspended, by sonication, in 10 mlNTGED-buffer +1% (v/v) Triton X-100. The mixture was centrifuged at25,000×g for 15 minutes and the supernatant discarded. The washedinclusion bodies were resuspended in 10 ml NTGED-buffer +0.1% (v/v)Triton X-100 and centrifuged at 25,000×g for 15 minutes. The wash wasrepeated with 10 ml NTGED-buffer+0.01% (v/v) Triton X-100 and thesuspension of inclusion bodies was aliquoted into 5 equal portions in 2niL vials prior to centrifugation in a Beckman Microfuge® 18 centrifugeat maximum speed. Supernatants were removed by pipetting and inclusionbodies were frozen at −20° C. until use.

[0257] Ni-NTA purification and IC5-derivatization of the β′,-fragment

[0258] The SDS-PAGE gel of the samples taken at different stages duringthe purification procedure can be seen in FIG. 15 (Coomassie-stain andIC5-sensitive scan). β′ inclusion bodies were resuspended in 3 mLTNTwGu-buffer+5 mM imidazole, incubated at RT for 15 minutes. Theprecipitate was spun down in a microfuge at 18,000 g (14,000 rpm) for 5minutes and the supernatant was loaded on a BioRad column (PolyPrep 10mL, 0.8×4 cm) with approximately 0.8 mL Ni-NTA matrix (Qiagen)previously equilibrated with 5 mL TNTwGu-buffer +5 mM imidazole. Toremove unbound protein, the column was washed with at least 3 mLTNTwGu-buffer +5 mM imidazole. To reduce any disulfide bonds, the columnwas washed with 5 mnL freshly prepared TNTwGu-buffer+20 mM imidazole and2 mM Tris(2-carboxyethyl)phosphine (TCEP). Excess TCEP andnon-specifically bound protein were removed by washing with 3 mLTNTwGu-buffer+20 mM imidazole which was saturated with N₂. The boundprotein was derivatized with IC5-maleimide by loading 2 niL freshlyprepared TNTwGu-buffer+20 mM imidazole and 0.2 mM IC5. The flow-throughwas reloaded onto the column twice before excess dye was removed bywashing with 3 niL TNTwGu-buffer+20 mM imidazole. Derivatized proteinwas eluted with TNTwGu-buffer+200 mM imidazole and stored denatured at−20° C.

[0259] Purification and derivatization of σ⁷⁰

[0260] One aliquot of inclusion bodies (10 nmol protein, 0.7 mg) wassolubilized by resuspending in 5 mL TGE-buffer+6 M GuHCl. To refoldproteins, the denaturant was diluted 100-fold by dripping into chilled500 mL TGE-buffer+0.01% Triton X-100 slowly stirring on ice. Ifprecipitation occurs, the precipitate is spun down in a centrifuge at25,000 g (15,000 rpm SS-34 rotor) for 15 minutes at 4° C. The refoldedprotein is then bound to an anion exchange resin by adding 1 g POROSHQ50 (PerSeptive Biosystems) dry resin as a suspension in 5 mLTGE-buffer+0.01% Triton X-100 directly into the mixture. After stirringfor 30 minutes the suspension is poured over a 25 mL Econo Pack column(BioRad) and washed with 5 mL NTG-buffer. The column was plugged, andthe resin was resuspended in 5 mL Storage-buffer, divided into 10 equalaliquots, transferred into empty rinsed Pharmacia spin-columns, andstored at −20° C. Prior to use, the buffer in the spin-columns wasremoved by centrifugation at 5000 g (4500 rpm) in a tabletop centrifuge.To label 67, 500 μL NTGE-buffer+0.01% Triton X-100 and 0.1 μmolDTPA-AMCA (a gift from the Heyduk lab) were used to resuspend the resinand incubated at RT for 30 minutes. The Eu-complex was formed by loading5 μM EuCl₃ onto the resin with the derivatized protein. Aftercentrifugation at 4500 rpm, the column was washed with 500 liLNTGE-buffer+0.01% Triton X-100. Then the labeled protein was eluted with100 μL NTGE-buffer+500 mM NaCl. The flow through (labeled 67°) was useddirectly in the assays.

[0261] The anion exchange resin POROS HQ50 (PerSeptive Biosystems) canbe exchanged for DE52 (Whatman). Furthermore, labeling can also be doneafter purification and elution from the ion-exchange column simply byadding the protein solution to an aliquot of the dye (same buffers andconditions). Excess label and Eu-ions were then removed by usingPharmacia G50 spin-columns.

[0262] Electrophoretic Mobility Shift (EMS) assay to test complexformation of labeled σ⁷⁰ and β′

[0263] The assays were performed in a buffer resulting of the use of2×Native Sample-buffer and NTG-buffer (200 μL total volume, 5% glycerol,50 mM Tris-HCl, pH 8.8, 50 mM NaCl, bromphenol blue 0.005% (w/v) and2.5% DMSO is used when insoluble additives are applied). The standardprotein concentrations were 2 μM 670 (labeled protein) and 2 μM β′(labeled protein) but can be lowered to 200 nM and 100 nM, respectively.Labeled 67 was added first to the assay, then the potential inhibitorand then denatured labeled β′. The solution was mixed well after theaddition of each component. The mixture was incubated for 5 minutes atRT, and then 15 liL were loaded on a precast polyacrylamide gel(12-well, 12%, Tris/glycine, NOVEX). The electrophoresis was conductedwith pre-chilled buffers, gels and apparatus in the cold room (4° C) atconstant voltage of 120 V (5-20 mA, variable) for 2.5 hours. TheIC5-emission was scanned on a Storm system (Molecular Dynamics) in thered fluorescence mode. The Eu emission was measured using an UV box(λ_(excit.)=312 nm, Fotodyne) with 6 seconds acquisition time. Totalprotein was stained with Coomassie blue stain using the Gel Codestaining solution (Pierce) according to the procedure of themanufacturer and scanned with a Hewlett Packard scanner using orangefilter settings.

[0264] FRET assay to test for the inhibition of protein-proteininteraction of labeled σ⁷⁰ and β′

[0265] The assay was performed in NTG-buffer (200 μL total volume) plus2.5% DMSO (when insoluble additives were used) with 10 nM σ^(70*)(labeled protein) and 50 nM β′* (labeled protein). Labeled σ⁷⁰ was addedfirst to the assay, then the potential inhibitor and denatured labeledβ′ was added last, mixing the solution well after the addition of eachcomponent. The mixture was incubated for 5 minutes at RT and measured ina 96-well plate (Costar 3650) with a multi-plate reader (Wallac,VictorV² 1420). For this time-resolved fluorescence measurement, themanufacturer's protocol (LANCE high count 615/665) was used (excitationwith 1000 flashes, at 325 nm, measurement was delayed by 100 μs andacquired for 50 μs at 615 and 665 nm). In fluorimetric measurements itis common to use a second emission wavelength as an internal standard.This allows correction for instrument noise, but also to normalize thesignal for the actual amount of donor in this particular case. This ispossible since donor and acceptor emission wavelengths are wellseparated and can be acquired separately with the multi-plate reader.The IC5-emission is corrected for the very small amount of signal fromthe Eu-emission band (by cross talk measurement of a standard) and thendivided by the intensity of the Eu signal. The normalization can beincluded into the overall measurement protocol and is described by themanufacturer of the multi-plate reader (Wallac). The nature of thismethod allows the differentiation of the loss of signal due to realinhibition from simple absorption caused by the inner filter effect fromthe substance. This assists in the identification of false positives inthe actual high-throughput screen.

[0266] Results

[0267] Electrophoretic Mobility Shift (EMS) assay to test for thecomplex formation of labeled σ⁷⁰ and β′

[0268] The EMS assays clearly showed that the labeled as well as theunlabeled proteins could form a complex that runs higher in the nativegel than 67 alone. The different scanning techniques also confirmed theidentity of the bands in the EMS assay (FIG. 11). Furthermore, the EMSassay showed that unlabeled β′-fragment can compete for the labeled 67°.Thus, unlabeled β′-fragment itself represents a positive control for anagent able to interfere with the binding of labeled β′-fragment and σ⁷⁰in the assay.

[0269] LRET assay to test for the inhibition of protein-proteininteraction of labeled σ⁷⁰ and β′

[0270] The LRET assay provides a fast and reproducible alternative tothe EMS assay to monitor the formation of a protein-protein interactionbetween labeled do and β′ as well as its inhibition. All results of theEMS assay were reproducible by the LRET assay, for example, competitionof labeled σ⁷⁰ binding to the β′-fragment by increasing amounts ofunlabeled do was observed (FIG. 17). In further experiments, thedependence of salt (NaCl) and solvents (DMSO) were characterized (FIGS.18 and 19). As can be seen for the influence of NaCl on the assay, thesalt concentration has a major effect on the signal, since it drops to50% when the NaCl concentration was increased from 100 to 400 mM. It isknown that this β′-fragment interaction with σ⁷⁰ is weaken by increasedNaCl concentration. On the other hand, DMSO, a potential solvent foreffectors to be tested, had no significant effect on the assay. As canbe seen in FIG. 19, the signal in the LRET assay is not criticallyaffected by the amount of DMSO present up to 5%. In the same experiment,ethanol showed a significant effect over the range of 1 to 5%. Thesignal-to-noise ratio for the assay was between 7 and 10. This isparticularly desirable for a high-throughput screen since it cuts downon false positives by more accurate readings.

[0271] Discussion

[0272] There are several reasons to believe that the primaryprotein-protein interaction between bacterial core RNA polymerase (RNAP)and sigma factors represents a prime target for drug discovery. The keyto the potential of this target is the absolute requirement of sigmabinding to core RNAP for the initiation of transcription; any bacterialcell will die upon uptake of an inhibitor that effectively blocks thisinteraction. In addition to a very high bioactivity, a good specificitycan also be expected since the binding region of both proteins is highlyconserved among bacteria (FIG. 20) and is significantly different fromany known eukaryotic analogue. This implies a very low probability forside effects to occur due to interference with human RNAP.

[0273] The site itself offers another advantage over many potential andspecific targets. Since the binding site on the β′-subunit of RNAPinteracts with all sigma factors of one bacterium, the development ofresistance via point mutations against an inhibitor which binds toeither one of them in the binding site is unlikely, since it would haveto occur in both A and all sigma factors at the same time. Due to therising incidence of antibiotic resistance and the growing need for newantibiotics, this has recently become a major issue in drug discovery.

[0274] Luminescence resonance energy transfer (LRET) to measure sigmabinding to core RNAP has been shown by Heyduk and coworkers to be aneffective and very sensitive method. To prepare a LRET donor in theassay described above, a well-characterized σ⁷⁰(442C) mutant was usedthat had all natural cysteine residues mutated to serine residues, whichwas derivatized with a DTPA-AMCA-maleimide Eu-complex. A fragment(residues 100-309) of the β′-subunit of RNAP with a N-terminal HMK-siteand His₆-tag fusion was derivatized with IC5-maleimide as LRET acceptor.The spectroscopic features of this pair of dyes and their behavior inLRET experiments are well characterized. Using EMS assays andspectrometric measurements using time-resolved fluorescence, it wasshown that the labeled proteins can bind to each other in allcombinations with and without the label. As controls, the unlabeledproteins were tested to determine if they could compete with theirlabeled counterpart. In both assays, EMS and LRET, unlabeled β′-fragmentor σ⁷⁰ were able to compete with their labeled counterpart for bindingin a complex. Therefore, the assay can be used to monitor d° toβ′-binding, and can be used to screen for inhibitors of thisprotein-protein interaction.

[0275] The assay represents a fast and sensitive probe for thisparticular complex-formation. Substrates and materials are eitherreadily available or can be prepared in a simple and efficientprocedures. All the labeled protein components showed excellentstability during storage, a great advantage when screening largelibraries with 10,000 to 100,000 or more substances. Furthermore, theLRET assay has a very high sensitivity so that measurements can beperformed at very low protein concentrations of 1 to 100 nM resulting ina very low cost per compound screened.

[0276] The LRET assay turned out to be not only very sensitive, but alsovery accurate. The very good signal-to-noise ratio of 7 to 10 and theinternal standard method that helps to distinguish binding inhibitionfrom fluorescence quenching by an inner filter effect of the testsubstance, contribute enormously to avoiding false positive readings.Also the very good compatibility of the assay with the use of DMSO, apotential solvent for test substances, adds to its applicability as ahigh-throughput screen. Since many natural products and most peptides orsmall molecules from combinatorial libraries have low solubility inwater, it is necessary to use organic solvents. In this respect, DMSOrepresents the most versatile and potent solvent and since it is oftenused in libraries, its compatibility is desirable for anyhigh-throughput assay.

[0277] Moreover, to improve the signal characteristics or just simply touse cheaper and more available compounds, probes other thanDTPA-AMCA-EuIII and IC5 which exhibit FRET-based signals can beemployed. A simple modification has been described above by exchangingIC5 (Dojindo, Japan) for Cy5 (Amersham) and has been shown to have noeffect on the assay. Other europium chelates like DTPA-cs124-R andTTHA-AMCA-R (Selvin, 1999) have been described to be suitable. Companieslike Packard ((Eu)K or XL665, TRACE reagents) as well as Wallac (DELFIAand LANCE reagents) are offering such dye pairs along with platereaders. For example, allophycocyanin APC (an analog of XL665)(Boisclair et al., 2000) can be used as an acceptor in combination witha Eu-donor. In all cases the dyes may be attached to specific antibodiesthat recognize the target proteins. Another rare earth metal, Terbium,can serve as an alternative LRET donor to Eu in similar complexes.Phalloidin-tetramethylrhodamine is also described as a LRET acceptor.Recently, different fusions of target proteins with variants of thegreen fluorescent protein have been used to monitor FRET in vivo (Harpuret al., 2001; Pollok and Heim, 1999) and could be employed to measurethe inhibitory effect of potential compounds under in vivo conditionswhich also takes into account the delivery of the drug to the targetorganism. For example, pairs selected from the following could beemployed: BFP, eGFP, CFP (cyan), YFP (yellow), and eYFP. Thus, proteinscould be delivered to any bacterium via transfection or conjugation oremployed in a yeast two hybrid system and used to screen potentialinhibitory compounds in vivo.

[0278] In the case of screening natural product libraries with ahigh-throughput assay, a positive hit could represent a new class ofantibiotic, since no substance is known with such a mode of action. Onthe other hand the screen will help to identify known antibiotics, forwhich the mode of activity has not yet been identified or that revealmore than one activity. Together with positive hits from combinatoriallibraries, these substances can then serve as lead structures to designand tailor a new compound with desirable characteristics such as: highactivity, specificity, stability and ability to enter the cell on theone hand and low side effects, costs, chance and likelihood ofresistance development on the other hand. In addition this assay canserve as a powerful tool to investigate the relative binding ofdifferent sigma factors and sigma factor mutants to core.

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[0378] All publications, patents and patent applications areincorporated herein by reference. While in the foregoing specification,this invention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

1 26 1 14 PRT Artificial Sequence An N-terminal tag 1 Met Ala Arg ArgAla Ser Val His His His His His His Met 1 5 10 2 13 PRT ArtificialSequence An N-terminal tag 2 Met Ala Arg Arg Ala Ser Val His His His HisHis His 1 5 10 3 13 PRT Artificial Sequence An N-terminal tag 3 Met AlaArg Arg Ala Ser Val His His His His His His 1 5 10 4 13 PRT ArtificialSequence An N-terminal tag 4 Met Arg Arg Ala Ser Val His His His His HisHis Ala 1 5 10 5 13 PRT Artificial Sequence An N-terminal tag 5 Met HisHis His His His His Ala Arg Arg Ala Ser Val 1 5 10 6 50 PRT Escherichiacoli 6 Phe Ala Thr Ser Asp Leu Asn Asp Leu Tyr Arg Arg Val Ile Asn Arg 15 10 15 Asn Asn Arg Leu Lys Arg Leu Leu Asp Leu Ala Ala Pro Asp Ile Ile20 25 30 Val Arg Asn Glu Lys Arg Met Leu Gln Glu Ala Val Asp Ala Leu Leu35 40 45 Asp Asn 50 7 50 PRT Thermus aquaticus 7 Phe Ala Thr Ser Asp LeuAsn Asp Leu Tyr Arg Arg Leu Ile Asn Arg 1 5 10 15 Asn Asn Arg Leu LysLys Leu Leu Ala Gln Gly Ala Pro Glu Ile Ile 20 25 30 Ile Arg Asn Glu LysArg Met Leu Gln Glu Ala Val Asp Ala Val Ile 35 40 45 Asp Asn 50 8 47 PRTEscherichia coli 8 Ala Lys Ala Arg Arg Ala Lys Lys Glu Met Val Glu AlaAsn Leu Arg 1 5 10 15 Leu Val Ile Ser Ile Ala Lys Lys Tyr Thr Asn ArgGly Leu Gln Phe 20 25 30 Leu Asp Leu Ile Gln Glu Gly Asn Ile Gly Leu MetLys Ala Val 35 40 45 9 47 PRT C. crescentus 9 Arg Glu Ala Arg Gln AlaLys Lys Glu Met Val Glu Ala Asn Leu Arg 1 5 10 15 Leu Val Ile Ser IleAla Lys Lys Tyr Thr Asn Arg Gly Leu Gln Phe 20 25 30 Leu Asp Leu Ile GlnGlu Gly Asn Ile Gly Leu Met Lys Ala Val 35 40 45 10 47 PRT P. putida 10Ala Lys Ala Arg Arg Ala Lys Lys Glu Met Val Glu Ala Asn Leu Arg 1 5 1015 Leu Val Ile Ser Ile Ala Lys Lys Tyr Thr Asn Arg Gly Leu Gln Phe 20 2530 Leu Asp Leu Ile Gln Glu Gly Asn Ile Gly Leu Met Lys Ala Val 35 40 4511 47 PRT H. influenza 11 Gln Lys Ala Arg Arg Ala Lys Lys Glu Met ValGlu Ala Asn Leu Arg 1 5 10 15 Leu Val Ile Ser Ile Ala Lys Lys Tyr ThrAsn Arg Gly Leu Gln Phe 20 25 30 Leu Asp Leu Ile Gln Glu Gly Asn Ile GlyLeu Met Lys Ala Val 35 40 45 12 47 PRT M. xanthus 12 Arg Arg Ala Glu ArgAla Lys Ser Glu Leu Val Glu Ala Asn Leu Arg 1 5 10 15 Leu Val Val SerIle Ala Lys Lys Tyr Thr Asn Arg Gly Leu Gln Phe 20 25 30 Leu Asp Leu IleGln Glu Gly Asn Ile Gly Leu Met Lys Ala Val 35 40 45 13 47 PRT S. aureus13 Gln Gly Asp Glu Val Ala Lys Ser Arg Leu Ala Glu Ala Asn Leu Arg 1 510 15 Leu Val Val Ser Ile Ala Lys Arg Tyr Val Gly Arg Gly Met Leu Phe 2025 30 Leu Asp Leu Ile Gln Glu Gly Asn Met Gly Leu Ile Lys Ala Val 35 4045 14 47 PRT B. subtilis 14 Glu Gly Asp Glu Glu Ser Lys Arg Arg Leu AlaGlu Ala Asn Leu Arg 1 5 10 15 Leu Val Val Ser Ile Ala Lys Arg Tyr ValGly Arg Gly Met Leu Phe 20 25 30 Leu Asp Leu Ile His Glu Gly Asn Met GlyLeu Met Lys Ala Val 35 40 45 15 47 PRT T. maritima 15 Met Gly Asp LysLys Ala Lys Glu Lys Leu Ile Thr Ser Asn Leu Arg 1 5 10 15 Leu Val ValSer Ile Ala Lys Arg Tyr Met Gly Arg Gly Leu Ser Phe 20 25 30 Gln Asp LeuIle Gln Glu Gly Asn Ile Gly Leu Leu Lys Ala Val 35 40 45 16 47 PRTEscherichia coli 16 Ala Lys Ala Arg Arg Ala Lys Lys Glu Met Val Glu AlaAsn Leu Arg 1 5 10 15 Leu Val Ile Ser Ile Ala Lys Lys Tyr Thr Asn ArgGly Leu Gln Phe 20 25 30 Leu Asp Leu Ile Gln Glu Gly Asn Ile Gly Leu MetLys Ala Val 35 40 45 17 44 PRT Escherichia coli 17 Val Ala Ser Arg ArgArg Met Ile Glu Ser Asn Leu Arg Leu Val Val 1 5 10 15 Lys Ile Ala ArgArg Tyr Gly Asn Arg Gly Leu Ala Leu Leu Asp Leu 20 25 30 Ile Glu Glu GlyAsn Leu Gly Leu Ile Arg Ala Val 35 40 18 44 PRT Escherichia coli 18 LeuGlu Ala Ala Lys Thr Leu Ile Leu Ser His Leu Arg Phe Val Val 1 5 10 15His Ile Ala Arg Asn Tyr Ala Gly Tyr Gly Leu Pro Gln Ala Asp Leu 20 25 30Ile Gln Glu Gly Asn Ile Gly Leu Met Lys Ala Val 35 40 19 37 PRTEscherichia coli 19 Tyr Val Pro Leu Val Arg His Glu Ala Leu Arg Leu GlnVal Arg Leu 1 5 10 15 Pro Ala Ser Val Glu Leu Asp Asp Leu Leu Gln AlaGly Gly Ile Gly 20 25 30 Leu Leu Asn Ala Val 35 20 50 PRT Escherichiacoli 20 Phe Ala Thr Ser Asp Leu Asn Asp Leu Tyr Arg Arg Val Ile Asn Arg1 5 10 15 Asn Asn Arg Leu Lys Arg Leu Leu Asp Leu Ala Ala Pro Asp IleIle 20 25 30 Val Arg Asn Glu Lys Arg Met Leu Gln Glu Ala Val Asp Ala LeuLeu 35 40 45 Asp Asn 50 21 50 PRT T. aquaticus 21 Phe Ala Thr Ser AspLeu Asn Asp Leu Tyr Arg Arg Leu Ile Asn Arg 1 5 10 15 Asn Asn Arg LeuLys Lys Leu Leu Ala Gln Gly Ala Pro Glu Ile Ile 20 25 30 Ile Arg Asn GluLys Arg Met Leu Gln Glu Ala Val Asp Ala Val Ile 35 40 45 Asp Asn 50 2250 PRT P. putida 22 Phe Ala Thr Ser Asp Leu Asn Asp Leu Tyr Arg Arg ValIle Asn Arg 1 5 10 15 Asn Asn Arg Leu Lys Arg Gln Leu Asp Leu Ser AlaPro Asp Ile Ile 20 25 30 Val Arg Asn Glu Lys Pro Met Leu Gln Glu Ala ValGlu Pro Leu Leu 35 40 45 Asp Asn 50 23 50 PRT H. influenza 23 Phe AlaThr Ser Asp Leu Asn Asp Leu Tyr Arg Arg Val Ile Asn Arg 1 5 10 15 AsnAsn Arg Leu Lys Arg Leu Leu Asp Leu Ile Ala Pro Asp Ile Ile 20 25 30 ValArg Asn Glu Lys Arg Met Leu Gln Glu Ser Val Asp Ala Leu Leu 35 40 45 AspAsn 50 24 50 PRT S. aureus 24 Phe Ala Thr Ser Asp Leu Asn Asp Leu TyrArg Arg Val Ile Asn Arg 1 5 10 15 Asn Asn Arg Leu Lys Arg Leu Leu AspLeu Gly Ala Pro Gly Ile Ile 20 25 30 Val Gln Asn Glu Lys Arg Met Leu GlnGlu Ala Val Asp Ala Leu Ile 35 40 45 Asp Asn 50 25 50 PRT B. subtilis 25Phe Ala Thr Ser Asp Leu Asn Asp Leu Tyr Arg Arg Val Ile Asn Arg 1 5 1015 Asn Asn Arg Leu Lys Arg Leu Leu Asp Leu Gly Ala Pro Ser Ile Ile 20 2530 Val Gln Asn Glu Lys Arg Met Leu Gln Glu Ala Val Asp Ala Leu Ile 35 4045 Asp Asn 50 26 50 PRT T. maritima 26 Phe Ala Thr Thr Asp Leu Asn GluLeu Tyr Arg Arg Leu Ile Asn Arg 1 5 10 15 Asn Asn Arg Leu Lys Lys LeuLeu Glu Leu Gly Ala Pro Glu Ile Ile 20 25 30 Leu Arg Asn Glu Lys Arg MetLeu Gln Glu Ala Val Asp Ala Leu Ile 35 40 45 His Asn 50

What is claimed is:
 1. A method to identify an agent which inhibits thebinding of σ of core RNA polymerase, a subunit thereof or a portionthereof, comprising: a) contacting the agent with core RNA polymerase,or an isolated subunit of RNA polymerase or a portion thereof, so as toform a complex; b) contacting the complex with σ or a portion thereof;and c) detecting or determining whether the agent inhibits or preventsthe binding of σ of core RNA polymerase or the isolated subunit of RNApolymerase or a portion thereof.
 2. A method to identify an agent whichinhibits the binding of σ of core RNA polymerase, a subunit thereof or aportion thereof, comprising: a) contacting the agent with σ or a portionthereof, so as to form a complex; b) contacting the complex with coreRNA polymerase, or an isolated subunit of RNA polymerase or a portionthereof; and c) detecting or determining whether the agent inhibits orprevents the binding of σ of core RNA polymerase or the isolated subunitof RNA polymerase or a portion thereof.
 3. The method of claim 1 or 2wherein the isolated subunit of RNA polymerase or a portion thereof isthe β′ subunit.
 4. The method of claim 3 wherein portion of the β′subunit comprises the interaction domain of the β′ subunit.
 5. Themethod of claim 4 wherein the portion of β′ comprises residues 270 to309 of β′, residues 265 to 309 of β′, residues 100 to 309 of β′, andresidues 260 to 309 of β′.
 6. The method of claim 1 or 2 wherein theisolated subunit is a fusion protein.
 7. The method of claim 1 or 2wherein the σ is a homologous σ.
 8. The method of claim 1 or 2 whereinthe σ is a heterologous σ.
 9. The method of claim 1 or 2 wherein thesubunit of RNA polymerase or a portion thereof has at least one aminoacid substitution relative to the native subunit.
 10. The method ofclaim 1 or 2 wherein core RNA polymerase, or an isolated subunit of RNApolymerase or a portion thereof is labeled or binds to a detectablelabel.
 11. The method of claim 10 wherein a is labeled or binds to adetectable label which is different than the label for core RNApolymerase, an isolated subunit thereof or a portion thereof.
 12. Themethod of claim 11 wherein β′ is labeled with IC5 and o is labeled withEu.
 13. The method of claim 1 or 2 wherein luminescence resonance energytransfer is employed to detect or determine whether the agent inhibitsor prevents binding of σ to core RNA polymerase, the isolated subunit ofRNA polymerase or a portion thereof.
 14. The method of claim 1 or 2wherein fluorescence resonance energy transfer is employed to detect ordetermine whether the agent inhibits or prevents binding of σ to coreRNA polymerase, the isolated subunit of RNA polymerase or a portionthereof.
 15. The method of claim 13 which employs europium or terbium.16. The method of claim 13 which employs IC5, Cy5, allophycocyanin APC,or phalloidin-tetramethylrhodamine.
 17. The method of claim 1 or 2wherein σ or a portion thereof is labeled or binds to a detectablelabel.
 18. The method of claim 17 wherein core RNA polymerase, or anisolated subunit of RNA polymerase or a portion thereof is labeled orbinds to a detectable label which is different than the label for σ or aportion thereof.
 19. An agent identified by the method of claim 1 or 2.20. A method to identify an agent which inhibits or prevents the bindingof σ to the β′ subunit of core RNA polymerase, comprising: a) contactinga prokaryotic cell with the agent; and b) detecting or determiningwhether the agent inhibits or prevents the binding of σ to the β′subunit of RNA polymerase in the cell.
 21. The method of claim 20wherein the cell encodes two different forms of the β′ subunit.
 22. Themethod of claim 21 wherein agent is contacted with the cell underconditions in which one of the forms is not expressed.
 23. The method ofclaim 22 wherein the form that is expressed has at least one amino acidsubstitution relative to the form which is not expressed.
 24. The methodof claim 20 wherein the inhibition of the growth of the cell is detectedor determined.
 25. The method of claim 20 wherein the agent inhibits orprevents the binding of a to residues 270 to 309 of 62 ′, residues 265to 309 of β′, residues 100 to 309 of β′ or residues 260 to 309 of β′subunit.
 26. An agent identified by the method of claim
 20. 27. A methodto inhibit the growth of a prokaryotic cell, comprising: contacting thecell with an effective amount of the agent of claim 19 or
 26. 28. A hostcell comprising a recombinant DNA molecule comprising a promoterfunctional in the host cell operably linked to a DNA segment encoding aβ′ subunit of RNA polymerase.
 29. The host cell of claim 28 wherein theDNA segment encodes a β′subunit having at least one amino acidsubstitution relative to the endogenous β′ subunit.
 30. The host cell ofclaim 28 wherein the DNA segment encodes a β′ subunit having aC-terminal deletion.
 31. The host cell of claim 28 wherein therecombinant DNA molecule encodes a fusion protein.
 32. An isolated andpurified portion of the β′ subunit of RNA polymerase which binds to σ invivo.
 33. The isolated and purified portion of the β′ subunit of claim32 which comprises a portion selected from the group consisting ofresidues 270 to 309, residues 265 to 309, residues 100 to 309 andresidues 260 to
 309. 34. A method to identify a region on a subunit ofcore RNA polymerase which specifically binds a, comprising: a)contacting core RNA polymerase, an isolated subunit or a portion thereofwhich comprises at least one amino acid substitution relative to nativecore RNA polymerase or a native subunit or portion thereof with σ or aportion thereof so as to form a complex; and b) detecting or determiningcomplex formation.
 35. The method of claim 34 wherein the substitutiondoes not inhibit complex formation.